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WO2024218019A1 - A method implementing morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and other related methods - Google Patents

A method implementing morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and other related methods Download PDF

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Publication number
WO2024218019A1
WO2024218019A1 PCT/EP2024/060083 EP2024060083W WO2024218019A1 WO 2024218019 A1 WO2024218019 A1 WO 2024218019A1 EP 2024060083 W EP2024060083 W EP 2024060083W WO 2024218019 A1 WO2024218019 A1 WO 2024218019A1
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Prior art keywords
organelle
extracellular
vesicle
ref
membrane
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French (fr)
Inventor
Abdou Rachid THIAM
Alexandre SANTINHO
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Centre National de la Recherche Scientifique CNRS
Sorbonne Universite
Universite Paris Sciences et Lettres
Universite Paris Cite
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Centre National de la Recherche Scientifique CNRS
Sorbonne Universite
Universite Paris Sciences et Lettres
Universite Paris Cite
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Definitions

  • the present invention relates to several methods which implement morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and/or to study the biological behaviour of membrane transport proteins.
  • the invention relates to the monitoring of intracellular ion channels, ion pumps and transporters activity thanks to morphological changes of extracellular organelle vesicles.
  • the present invention includes an in vitro or ex vivo method for examining the transporting activity and/or studying the biological behaviour of at least one membrane transport protein with respect to at least one specific compound, an in vitro or ex vivo method to determine the presence of at least one membrane transport protein in at least one extracellular organelle vesicle of a specific type, an in vivo or ex vivo method for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein, an in vitro or ex vivo method for determining abnormal expression and/or abnormal transporting activity of at least one membrane transport protein, and an in vitro or ex vivo method for evaluating the response of at least one membrane transport protein contained in a membrane of an extracellular organelle vesicle with respect to a response of said membrane transport protein contained in a cell component.
  • Transport membrane proteins such as ion channels represent an essential pharmacological target for drugs such as painkillers, anticonvulsants and neuromuscular blockers.
  • the hit discovery rate of drugs for channels is severely impeded by several parameters: accessibility of the ion channel in a relevant membrane environment, and the cost and robustness of the current readout methods.
  • most ion channels/pumps studied so far are localized at the plasma membrane. The membrane delineates the cell and limits access to intracellular ion channels/pumps.
  • heavy and costly methods are currently implemented by pharmaceuticals to measure the activity of ion fluxes across pumps/channels to develop drugs against these.
  • HTS High Throughput Screening
  • the present inventors have found fast and reliable method for analysis of transport membrane protein activity.
  • Said method allows for example the discovery of molecules that can modulate the activity of intracellular transport membrane proteins channeling passage of ions or solutes across membranes. Additionally, a lot of ion channels are crucial for our health, and, if some drug candidate alters their activity, it can lead to toxicity.
  • the method of the present invention which allows the reading of ion channels activity can serve as efficacy tests but also for toxicity tests.
  • the method of the present invention is focused on quantifying the size/morphology change, and/or luminal fluorescence and/or surface membrane tension of specific extracellular organelle vesicles so as to examine the activity of a transport membrane protein with respect to a given molecule.
  • a first object of the present invention is an in vitro or ex vivo method for examining the transporting activity and/or studying the biological behaviour of at least one membrane transport protein with respect to at least one compound C, wherein said method comprises at least the following steps: i) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, ii) contacting said compound C with said extracellular organelle vesicle, and iii) maintaining said compound C and said extracellular organelle vesicle into contact, wherein said method further comprises:
  • compound C can also be defined as a molecule likely to be transported by a membrane transport protein.
  • Organelles used in the method of the present invention are biological material initially present in cells that are extracted from said cells in the form of vesicles. Such organelles are therefore outside of their hosting cell (hence the name "extracellular”) in the form of vesicles before implementing the method of the present invention.
  • the method of the present invention does not require the extraction or isolation of membrane transport proteins, which are localized in their original membrane and in their original organelle.
  • the membrane transport proteins examined in the method of the present invention are only extracted from their hosting cell while maintaining their integrity.
  • the method of the invention is a method which avoids the denaturation, extraction and/or isolation of membrane transport proteins and which makes it possible to study them in their "native" environment, i.e. in their hosting organelle vesicle. This is totally different from methods of the prior art which implement reconstitution of membrane transport proteins in artificial models based on DIB ("droplet-interface bilayer"), SUV ("small unilamellar vesicle”) and/or GUV (“giant unilamellar vesicle”).
  • DIB droplet-interface bilayer
  • SUV small unilamellar vesicle
  • GUV giant unilamellar vesicle
  • the extracellular organelle vesicles can be extracted from mammalian, plant and/or bacteria cells, and preferably extracted from mammalian cells.
  • Examples of such cells include COS-7 cells, HeLa cells, HEK cells, CHO cells, fibroblast cells, red blood cells, T-cells, neuroblastoma cells, stem cells, iPSC (induced Pluripotent Stem Cells) derived into cell types such iPSC derived neuronal stem cells, iPSC derived mesenchymal stem cells, iPSC derived monocytes stem cells, iPSC derived cardiomyocyte stem cells, iPSC derived microglia stem cells, iPSC derived myotubes stem cells, etc ...
  • iPSC induced Pluripotent Stem Cells
  • Said cells can be obtained from the market, from laboratories and/or from hospitals, and/or recovered from organoids, biopsies, tissues, organs and/or organisms, from healthy or diseased human patient origin.
  • the morphological change(s) can occur between step i) and step iii), between step ii) and step iii), or both between step i) and step iii) and between step ii) and step iii).
  • the comparison is generally made between corresponding (or same) features.
  • a morphological change occurring between step i) and step iii) is compared to a reference morphological change occurring between step i) and step iii), etc...
  • Observation of morphological change(s) of said extracellular organelle vesicle can be carried out by means such as confocal microscopy.
  • fluorescence variation can be analysed simultaneously with high throughput screening machine for imaging such as Molecular Devices, from Hamatsu's et al.
  • Morphological changes can be selected from deflation and inflation (also called re-swelling).
  • Said morphological change(s) of said extracellular organelle vesicle between step i) and step iii) and/or between step ii) and step iii) can be compared with morphological changes occurring with a same method where one parameter has been modified such as with a different compound instead of compound C, with addition of a new compound such as a drug candidate, with an extracellular organelle vesicle of the same type but from a different cell, with an extracellular organelle vesicle of a different type, for example from a same or different cell, or with one or more cellular components, for example from a same or different cell.
  • Said membrane transport protein is contained in at least one of said membrane or a membrane of a reference extracellular organelle vesicle.
  • said membrane transport protein is contained in said membrane, in a membrane of a reference extracellular organelle vesicle, or both in said membrane and in a membrane of a reference extracellular organelle vesicle.
  • Said morphological change(s) results in a change in at least one parameter, preferably selected from a volume V (in pm 3 ), a membrane surface tension T (in N/m), and a luminal fluorescence value F (in arbitrary unit).
  • the volume V relates to the volume of extracellular organelle vesicle
  • the membrane surface tension T relates to the membrane surface tension T of the membrane of the extracellular organelle vesicle
  • the luminal fluorescence value F relates to the luminal fluorescence value F of the intravesicular aqueous medium.
  • the luminal fluorescence value F can be obtained thanks to grafting or labelling a resident protein of the extracellular organelle vesicle (as explained later in the processes to obtain said extracellular organelle vesicles).
  • the luminal fluorescence is by definition fluorescence of lumen.
  • the luminal fluorescence can be obtained by labelling luminal proteins or luminal metabolites.
  • the luminal fluorescence has therefore to be distinguished from fluorophore reporting the concentration of compound C which can be obtained by using molecules that complex with compound C and emit fluorescence when interacting with compound C (Ex : FURA2, Fluo4, Fluo8).
  • Such fluorescence of compound C does not corresponds to a parameter characterizing the extracellular organelle vesicle, and even less to a parameter characterizing the morphological change of said extracellular organelle vesicle.
  • the luminal fluorescence contrary to the fluorescence of compound C, is independent from the concentration of compound C.
  • the labelling of compound C (with at least one fluorophore molecule reporting for the compound C) can be performed as an additional tool as explained in example 3.
  • the method can be carried with one or more extracellular organelle vesicles.
  • Step i) can implement several extracellular organelle vesicles of the same type or several extracellular organelle vesicles of different types, such as for example of at least two different types.
  • step i) said extravesicular aqueous medium and said intravesicular aqueous medium are preferably in a state of equilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have identical or almost identical osmolarities (in mOsm/L).
  • the term "almost identical" means that a difference of at most 20%, and preferably at most 10%, can be found between both osmolarities.
  • the contacting step ii) leads to a state of desequilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have different osmolarities (in mOsm/L).
  • the contact maintaining step iii) leads to a return to equilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have identical or almost identical osmolarities (in mOsm/L).
  • Step iii) is preferably carried out during at least 10 seconds.
  • the method according to the first object of the present invention comprises at least the following steps: i) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, and said extracellular organelle vesicle being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm 3 ), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii) contacting said compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm 3 ), a membrane surface tension Tn, and a luminal fluorescence value Fii (in arbitrary unit), iii) maintaining said compound C and said extracellular organelle vesicle into contact
  • step iv) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle.
  • other means can be used.
  • the comparison is generally made between corresponding (or same) features.
  • corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf, re f/Vii,ref, or Vf/Vi is compared with Vf, re f/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf, re f/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff, re f/Fi,ref.
  • transporting activity of said membrane transport protein with respect to said compound C is found when at least one of the following equation is verified:
  • a rate of transporting activity (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method with a first reference compound instead of said compound C, said membrane transport protein not having a transporting activity with respect to said first reference compound and the second corresponding reference ratio is obtained by applying said method with a second reference compound instead of said compound C, said membrane transport protein having a known good transporting activity with respect to said second reference compound.
  • the measuring in step ii) [respectively the measuring in step Hi)] can be dynamic or static.
  • the measurement in step ii) [respectively in step Hi)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
  • step ii) is not required.
  • the method according to the first object of the present invention is a method for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein with respect to at least one compound C, wherein it comprises at least the following steps: a) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, and said extracellular organelle vesicle being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm 3 ), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), b) contacting said compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm 3 ), a membrane surface tension Tii (in N/m
  • step e) The calculation of a ratio as defined in step e) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle.
  • other means can be used.
  • the comparison is generally made between corresponding (or same) features.
  • corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf, re f/Vii,ref, or Vf/Vi is compared with Vf, re f/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf, re f/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff, re f/Fi,ref.
  • inhibitory effect of said candidate compound is found when at least one of the following equation is verified:
  • a rate of inhibitory activity (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method without the presence of said candidate compound and the second corresponding reference ratio is obtained by applying said method with a reference candidate compound instead of said candidate compound, said reference candidate compound having a known good inhibitory effect on the transporting activity of said membrane transport protein with respect to said compound C.
  • the measuring in step b) [respectively the measuring in step d)] can be dynamic or static.
  • the measurement in step b) [respectively in step d)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
  • step b) the measuring in step b) is not required.
  • the contacting step c) is preferably carried out with a solution (e.g. aqueous solution) comprising said candidate compound at a molar concentration ranging from 0.001 pM to 100 mM, and preferably ranging from 0.1 nM to 100 pM.
  • a solution e.g. aqueous solution
  • the contacting step c) is preferably performed during at least 5 seconds, and more preferably during at least 30 seconds.
  • the method according to the second embodiment can be carried out with extracellular organelle vesicles of different types so as to evaluate toxicity of said candidate compound with respect to some of said different types of extracellular organelle vesicles.
  • the method according to the first object of the present invention is a method for determining abnormal expression and/or abnormal transporting activity of at least one membrane transport protein with respect to at least one compound C, comprising at least the following steps:
  • steps A) to D)] to a reference extracellular organelle vesicle from cells of an healthy patient or from non-genetically-modified cells, and wherein said membrane transport protein has transporting activity with respect to said compound C in said cells of a healthy patient or in said non-genetically-modified cells.
  • the method can be implemented to diagnose (/n vivo and/or ex vivo diagnostic) a disease affecting said patient, said disease being characterized by abnormal expression and/or abnormal transporting activity of said at least one membrane transport protein with respect to said compound C.
  • the calculation of a ratio as defined in step D) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle.
  • other means can be used.
  • the comparison is generally made between corresponding (or same) features.
  • corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf, re f/Vii,ref, or Vf/Vi is compared with Vf, re f/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf, re f/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff, re f/Fi,ref.
  • overexpression of said membrane transport protein is found when at least one of the following equation is verified:
  • a rate of altered transport protein activity of a patient from a disease related to said membrane transport protein (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method to a first reference extracellular organelle vesicle from cells of an healthy patient and the second corresponding reference ratio is obtained by applying said method to a second reference extracellular organelle vesicle from cells of an sick patient.
  • the measuring in step B) [respectively the measuring in step C)] can be dynamic or static.
  • the measurement in step B) [respectively in step C)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
  • step B) the measuring in step B) is not required.
  • the method of the first object of the present invention is a method to determine the presence of at least one membrane transport protein in at least one extracellular organelle vesicle of type 1, wherein said method comprises at least the following steps: i') providing in an extravesicular aqueous medium at least one extracellular organelle vesicle of type 1, said extracellular organelle vesicle of type 1 comprising a membrane surrounding an intravesicular aqueous medium, said extracellular organelle vesicle of type 1 being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm 3 ), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii') contacting at least one compound C with said extracellular organelle vesicle of type 1, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle of type 1 selected from a volume Vn (in
  • step iv' The calculation of a ratio as defined in step iv') is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle.
  • other means can be used.
  • the comparison is generally made between corresponding (or same) features.
  • corresponding ratios are compared. More particularly, f/ ii is compared with Vf, re f/Vii,ref, or f/ i is compared with Vf, re f/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf, re f/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff, re f/Fi,ref.
  • the measuring in step ii') [respectively the measuring in step Hi')] can be dynamic or static.
  • the measurement in step ii') [respectively in step Hi')] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
  • step ii' the measuring in step ii' is not required.
  • the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 can come from a same cell or a different cell.
  • the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 can be present (or comprised) in said extravesicular aqueous medium of step i '), with physical contact or without physical contact.
  • the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 come from a same cell, they are in physical contact in said extravesicular aqueous medium of step i').
  • the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 come from a different cell, they may be or not be in physical contact in said extravesicular aqueous medium of step i').
  • the method according to the fourth embodiment can be used to determine the activity of a membrane transport protein which is present in two different types of organelles but without the same density and/or without the same membrane environment.
  • the method of the first object of the present invention is a method to evaluate the response of at least one membrane transport protein contained in a membrane of an extracellular organelle vesicle with respect to a response of said membrane transport protein contained in a cell component, wherein said method comprises at least the following steps:
  • IV' calculating at least one ratio selected from Vf/Vii, Tf/Tii, Ff/Fu, Vf/Vi, Tf/Ti, and Ff/Fi,
  • V' comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff,ref/Fil,ref, Vf, ref/ Vi, ref, Tf,ref/Ti,ref, a nd Ff,ref/Fi,ref, wherein the corresponding reference ratio is obtained:
  • step IV' The calculation of a ratio as defined in step IV') is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a cellular component.
  • other means can be used.
  • the comparison is generally made between corresponding (or same) features.
  • corresponding ratios are compared. More particularly, f/ ii is compared with Vf, re f/Vii,ref, or f/ i is compared with Vf, re f/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf, re f/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff, re f/Fi,ref.
  • the measuring in step II') [respectively the measuring in step III')] can be dynamic or static.
  • the measurement in step II') [respectively in step III')] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
  • step III' the measuring in step III' is not required.
  • the cell component and the extracellular organelle vesicle can come from a same cell or a different cell.
  • the cell component and the extracellular organelle vesicle can be present (or comprised) in said extravesicular aqueous medium of step I'), with physical contact or without physical contact.
  • step I' when the cell component and the extracellular organelle vesicle come from a same cell, they are in physical contact in said extravesicular aqueous medium of step I'). When the cell component and the extracellular organelle vesicle come from a different cell, they may be or not be in physical contact in said extravesicular aqueous medium of step I').
  • the extracellular organelle vesicle in the method according to the first, second, third and fifth embodiments of the first object of the present invention is selected from organelles of endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, and multivesicular bodies, preferably is an extracellular organelle vesicle of Golgi apparatus, endoplasmic reticulum, mitochondria, lysosome, or endolysosome, and more preferably of endoplasmic reticulum.
  • the extracellular organelle vesicle of type 1 (respectively the extracellular organelle vesicle of reference type) in the method according to the fourth embodiment of the first object of the present invention is selected from organelles of endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, and multivesicular bodies.
  • the method according to the fourth embodiment of the first object of the present invention is implemented with several types of extracellular organelle vesicles, preferably from a same type of cell, so as to screen which type(s) of organelle vesicles has said at least one membrane transport protein as a resident protein, said several types of extracellular organelle vesicles being preferably selected from the following types: endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, multivesicular bodies.
  • the extracellular organelle vesicle of type 1 can be a extracellular mitochondria vesicle; and the extracellular organelle vesicle of reference type can be a extracellular endoplasmic reticulum vesicle.
  • the cell component is selected from cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, and plasma membrane, and preferably selected from cell nucleus and plasma membrane.
  • the cell component can be genetically enriched with said membrane transport protein.
  • the membrane transport protein is overexpressedin said cell component.
  • the membrane transport protein is preferably an ion channel, an ion pump, or a transporter, and more preferably an ion channel or an ion pump.
  • ion pumps include Lysosomal proton pumps, Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) pumps, Endoplasmic reticulum Ca 2+ ATPase (ERCA) pumps, P-type ATPase pumps, Na + /K + ATPase pumps, Ca 2+ /Mn 2+ ATfPase pumps, Proton ATPase pumps, V-ATPase, Sodium/potassium-ATPase (Na + /K + -ATPase), H + ATPase, P-type ATPase, Mg 2+ ATPase, Ca 2+ ATPase, ATP13A2/PARK9, preferably Lysosomal proton pumps, Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) pumps, Endoplasmic reticulum Ca 2+ ATPase (ERCA) pumps, Na + /K +
  • ion channels include Inositol trisphosphate (IP3) receptors, Ryanodine receptors (RyRs), Lysosomal anion channel CLC-7, TMEM175 H + channel, TMEM Channels, Transient receptor potential mucolipin 1 (TRPML1), Transient receptor potential mucolipin 2 (TRPML2), Transient receptor potential mucolipin 3 (TRPML3), Chloride channels CliC, Anion-selective channel 1 (ASCL1), Acid-sensing ion channels (ASICs), G-protein-gated acid-sensing ion channel (ASIC-like, GAC), Mitochondrial ATP-sensitive potassium (mKATP) channels, Mitochondrial voltagedependent anion-selective channel (VDAC), Calcium release-activated calcium (CRAC) channels, Store-operated calcium (SOC) channels, Inwardly rectifying potassium (Kir) channels, ATP-sensitive potassium (KATP) channels, Inwardly rectifying potassium (
  • transporters examples include Lysosomal amino acid transporter 1 (LAT1), Lysosomal amino acid transporter 2 (LAT2), Cationic amino acid transporter 3 (CAT3), Lysosomal chloride transporter CLCN7, Lysosomal cystine transporter cystinosin, Lysosomal iron transporter NRAMP2, Lysosomal cholesterol transporter NPC1, Lysosomal sphingolipid transporter NPC2, Lysosomal glucose transporter GLUT5, Lysosomal glucose transporter GLUT7, Lysosomal fructose transporter
  • LAT1 Lysosomal amino acid transporter 1
  • LAT2 Lysosomal amino acid transporter 2
  • CAT3 Cationic amino acid transporter 3
  • Lysosomal chloride transporter CLCN7 Lysosomal cystine transporter cystinosin
  • Lysosomal iron transporter NRAMP2 Lysosomal cholesterol transporter NPC1, Lysosomal sphingolipid transporter
  • Lysosomal fatty acid transporter CD36 Lysosomal fatty acid transporter LIMP-2, Lysosomal folate transporter FOLR1, Lysosomal vitamin C transporter SVCT2, Lysosomal zinc transporter ZIP4, Lysosomal copper transporter CTR1, Lysosomal glutathione transporter ABCG2, Lysosomal lipid transporter MLN64, Lysosomal drug transporter ABCB1, Lysosomal drug transporter ABCG2, Lysosomal drug transporter SLC22A16, Lysosomal drug transporter SLC22A17, Sodium-dependent phosphate transporter 2b (NaPi2b), Proton-coup
  • SLC22A5/organic cation transporter SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter, SLC22All/organic anion transporter, Mitochondrial phosphate transporter (PiC),
  • Mitochondrial carnitine/acylcarnitine transporter CACT
  • Mitochondrial malate transporter DIC
  • Mitochondrial adenosine nucleotide transporter ANT
  • Mitochondrial citrate transporter CiC
  • Mitochondrial folate transporter MFT
  • Sodium-dependent phosphate transporter 2b NaPi2b
  • Proton-coupled folate transporter PCFT
  • Zinc transporter 3 Zinc transporter 3 (ZnT3)
  • Copper transporter 1 CTR1
  • Na + /HCO3’ cotransporter 3 NBC3
  • Na + /dicarboxylate cotransporter 1 NaDCl
  • SLC26A3/dicarboxylate transporter SLC26A6/sulfate transporter
  • SLC26A7/chloride transporter Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides
  • SLC22 All/organic anion transporter, Lysosomal amino acid transporter 1 (LAT1), Lysosomal amino acid transporter 2 (LAT2), Cationic amino acid transporter 3 (CAT3), Lysosomal chloride transporter CLCN7, Lysosomal cystine transporter cystinosin, Lysosomal iron transporter NRAMP2, Lysosomal cholesterol transporter NPC1, Lysosomal sphingolipid transporter NPC2, Lysosomal glucose transporter GLUT5, Lysosomal glucose transporter GLUT7, Lysosomal fructose transporter
  • SLC22A5/organic cation transporter SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter, SLC22All/organic anion transporter, Mitochondrial phosphate transporter (PiC),
  • Mitochondrial carnitine/acylcarnitine transporter CACT
  • Mitochondrial malate transporter DIC
  • Mitochondrial adenosine nucleotide transporter ANT
  • Mitochondrial citrate transporter CiC
  • Mitochondrial folate transporter MFT
  • Sodium-dependent phosphate transporter 2b NaPi2b
  • Proton-coupled folate transporter PCFT
  • Zinc transporter 3 Zinc transporter 3 (ZnT3)
  • Copper transporter 1 CTR1
  • Na + /HCO3' cotransporter 3 NBC3
  • Na + /dicarboxylate cotransporter 1 NaDCl
  • SLC26A3/dicarboxylate transporter SLC26A6/sulfate transporter
  • SLC26A7/chloride transporter Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides
  • SER.CA ion pumps of endoplasmic reticulum and TMEM and TPC lysosomal channels are most preferred examples.
  • the compound C is preferably selected from ions, cellular metabolites, pyruvate, amino acids, fatty acids, fatty acids CoA, malate, citrate, isocitrate, and vitamins, and more preferably selected from ions.
  • Preferred examples of ions as compound C are Na + , K + , Ca 2+ , H + , HPC 2 ', HCCh' , Mg 2+ , Cl', Fe 2+ , Fe 3+ and other ions generally present in a cell cytoplasm or in an organelle lumen, more preferably Na + , K + , Ca 2+ , H + , HPC 2 ', HCOs' , Mg 2+ , Cl', Fe 2+ , Fe 3+ , and even more preferably Ca 2+ .
  • the membrane transport protein is an ion channel or an ion pump and the compound C is selected from ions, and preferably selected from Na + , K + , Ca 2+ , H + , HPC 2 ', HCCh' , Mg 2+ , Cl', Fe 2+ , Fe 3+ and other ions generally present in a cell cytoplasm or in an organelle lumen.
  • the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endoplasmic reticulum vesicle
  • the compound C is Ca 2+
  • the membrane transport protein is a Sarco/endoplasmic reticulum Ca 2+ ATPase (SER.CA) pump or the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endolysosome vesicle or extracellular lysosome vesicle
  • the compound C is K +
  • the membrane transport protein is a TMEM175 ion channel
  • the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endoplasmic reticulum vesicle
  • the compound C is Ca 2+
  • the membrane transport protein is a
  • the extracellular organelle vesicle of type 1 (respectively the extracellular organelle vesicle of reference type) in the method according to the fourth embodiment of the first object of the present invention is an extracellular endoplasmic reticulum vesicle, the compound C is Ca 2+ , and the membrane transport protein is a Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) pump.
  • SERCA Sarco/endoplasmic reticulum Ca 2+ ATPase
  • the extravesicular aqueous medium has preferably an osmolarity OSext ranging from 0.1 to 600 mOsm/L, and preferably from 0.1 to 100 mOsm/L, before the contacting step with said compound C (i.e. before contacting step ii), ii'), II'), b), B)).
  • the intravesicular aqueous medium has preferably an osmolarity OSint ranging from 0.1 to 600 mOsm/L, and more preferably from 0.1 to 100 mOsm/L, before the contacting step with said compound C (i.e. before contacting step ii), ii'), II', b), B)).
  • the contacting step i.e. contacting step ii), ii'), II'), b), B)
  • contact maintaining step i.e. contact maintaining step iii), iii'), III'), d), C)
  • the contacting step i.e. contacting step ii), iii'), II'), b), B)
  • contact maintaining step i.e. contact maintaining step iii), iii'), III'), d), C)
  • the contacting step (i.e. contacting step ii), ii'), II'), b), B)) is preferably carried out with a solution (more preferably an aqueous solution) comprising said compound C at a molar concentration ranging from 0.001 pM to 300 mM, and preferably ranging from 0.01 pM to 10 mM.
  • step ii) (respectively step ii), ii'), II'), b), B)) is performed by contacting a solution of said compound C with said extracellular organelle vesicle.
  • the solution comprising said compound C can be an aqueous solution such as a buffer solution, preferably having an osmolarity between 0.1 mOsm/L to 600 mOsm/L, and more preferably having an osmolarity between 0.1 mOsm/L to 100 mOsm/L.
  • the solution comprising said compound C is different from a hydrophobic solution such as an oily phase.
  • the extracellular organelle vesicle (respectively the extracellular organelle vesicle of reference type) (respectively the extracellular organelle vesicle of type 1) has preferably a lumen, is bilayer-bounded and is not surrounded by plasma membrane of a hosting cell.
  • the method according to the first object of the present invention further comprises during contacting step (i.e. contacting step ii), ii'), II'), b), B)) and contact maintaining step (i.e. contact maintaining step iii), iii'), III'), d), C)), at least one patch-clamp step so as to measure an electric current between the extracellular and intravesicular aqueous media.
  • the method further comprises adding at least one fluorophore molecule reporting for the compound C to the intravesicular aqueous medium so that during contacting step (i.e. contacting step ii), ii'), III'), b), B)) and contact maintaining step (i.e. contact maintaining step iii), iii'), III'), d), C)) a volumic concentration of the compound C in the intravesicular aqueous medium is measured.
  • the fluorophore molecule is added before the contacting step with compound C (i.e. before contacting step ii), ii'), II'), b), B)), such as for example on the cells before extraction of the organelles or after extraction.
  • This fluorophore molecule is permeable to membranes so that it is already present in the extracellular organelle vesicle before the measurement process.
  • the fluorophore molecule becomes fluorescent because its fluorescence is proportional to the intravesicular concentration of compound C.
  • the method further comprises adding at least one biological modulator (also called biomodulator) reporting for the membrane transport protein, preferably to the intravesicular aqueous medium.
  • at least one biological modulator also called biomodulator
  • the biological modulator favors or regulates the functioning of the membrane transport protein.
  • the biological modulator is proper to each membrane transport protein.
  • the biological modulator is ATP when the membrane transport protein is a Sarco/endoplasmic reticulum Ca 2+ ATPase (SER.CA) pump.
  • SER.CA Sarco/endoplasmic reticulum Ca 2+ ATPase
  • biological modulators can include GTP, UTP, CTP, binding proteins such as G proteins, AMPc, Phospholamban, calmodulin, protein kinase A, protein kinase C, protein kinase G, protein kinase D, protein phosphatases such as PPI or PP2A, CaMKII, phosphoinositides, Nitric Oxide, PLC, cAMP, and cGMP.
  • Said biological modulators are well-known by a person skilled in the art and the person skilled in the art can select the appropriate biological modulator depending on the membrane transport protein implemented in the method of the present invention.
  • the biological modulator is added simultaneously with compound C (i.e. during contacting step ii), ii'), II'), b), B)).
  • step ii) (respectively step ii), ii'), II'), b), B)) is performed by contacting a solution comprising said compound C and said biological modulator, with said extracellular organelle vesicle.
  • the solution comprising said compound C and said biological modulator can be an aqueous solution such as a buffer solution, preferably having an osmolarity between 0.1 mOsm/L to 600 mOsm/L, and more preferably having an osmolarity between 0.1 mOsm/L to 100 mOsm/L.
  • a buffer solution preferably having an osmolarity between 0.1 mOsm/L to 600 mOsm/L, and more preferably having an osmolarity between 0.1 mOsm/L to 100 mOsm/L.
  • the solution comprising said compound C and said biological modulator is different from a hydrophobic solution such as an oily phase.
  • At least one of the contacting step or the contact maintaining step leads to morphological change(s) of said extracellular organelle vesicle, and preferably to a deflation of said extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contacting step (i.e. contacting step ii), II'), b), B); respectively ii')) followed by a reswelling of the extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contact maintaining step (i.e. contact maintaining step iii), III'), d), C); respectively iii')).
  • morphological changes are indicative of a good transporting activity of said membrane transport protein with respect to compound C (i.e. healthy patient, normal expression, promoter effect of the candidate compound, presence/good specifity of the membrane transport protein and/or good response of the membrane transport protein).
  • At least one of the contacting step or the contact maintaining step leads to morphological change(s) of said extracellular organelle vesicle, and preferably to a deflation of said extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contacting step (i.e. contacting step ii), II'), b), B), respectively ii')) followed by no reswelling of the extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contact maintaining step (i.e. contact maintaining step iii), III'), d), C), respectively iii')).
  • morphological changes are indicative of a bad transporting activity of said membrane transport protein with respect to compound C (i.e. non-healthy patient, abnormal expression, inhibitor effect of the candidate compound, absence/bad specifity of the membrane transport protein and/or bad response of the membrane transport protein).
  • the method further comprises before step i), a), A), I') or i '), a step of adding at least an amount of said compound C into the intravesicular aqueous medium of the extracellular organelle vesicle (loading step).
  • step i When compound C is an ion, such step can be called an ion loading step. Thanks to this loading step, the extracellular organelle vesicle is loaded with compound C and in particular specific ions.
  • Said loading step can be carried out:
  • the method according to the first object of the present invention implements extracellular organelle vesicles.
  • the extracellular organelle vesicles allow a fast quantification of drug interaction and/or effect of one or more compounds on transport membrane protein activity. They facilitate the method of the present invention compared to other techniques from the art which have to concentrate the fragments of organelles and then do some biochemistry quantification.
  • the claimed method allow to study organelles interactions between cellular components in a new way and the role of intracellular proteins and lipids.
  • extracellular organelle vesicles also called “giant extracellular organelle vesicles” or GEOVs
  • GEOVs giant extracellular organelle vesicles
  • the extracellular organelle vesicles can be extracted from mammalian, plant and/or bacteria cells, and preferably extracted from mammalian cells.
  • Examples of such cells include COS-7 cells, HeLa cells, HEK cells, CHO cells, fibroblast cells, red blood cells, T-cells, neuroblastoma cells, stem cells, iPSC (induced Pluripotent Stem Cells) derived into cell types such iPSC derived neuronal stem cells, iPSC derived mesenchymal stem cells, iPSC derived monocytes stem cells, iPSC derived cardiomyocyte stem cells, iPSC derived microglia stem cells, iPSC derived myotubes stem cells, etc ...
  • Said cells can be obtained from the market, from laboratories and/or from hospitals, and/or recovered from organoids, biopsies, tissues, organs and/or organisms, from healthy or diseased human patient origin.
  • Said extracellular organelle vesicles can have a mean surface-to-volume ratio S/V (surface divided by the volume of a geometric shape, spherical in most of the case) ranging from 3 pm’ 1 to 0.15 pm’ 1 , preferably from 2 pm to 0.15 pm , more preferably from 1.5 pm -1 to 0.15 pm 4 , the most preferably between 1.2 pm 4 to 0.15 pm 4 . It generally corresponds to a mean size ranging from about 2 to 40 pm, preferably from 3 to 20 pm, more preferably from 4 to 15 pm, the most preferably from 5 to 10 pm.
  • S/V surface divided by the volume of a geometric shape, spherical in most of the case
  • said extracellular organelle vesicles have an increased surface- to-volume ratio which generally ranges from 1.2 pm 4 (for a size > 5 pm) to 0.3 pm -1 (for a size ⁇ 20 pm).
  • Such extracellular organelle vesicles preferably derive from organelles from the group consisting of endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, multivesicular bodies.
  • both membrane and lumen of such extracellular organelle vesicles are composed of proteins, lipids and metabolites.
  • the total amount of biochemical material of such extracellular organelle vesicles can be a fraction of the total composition of the organelle from which they are produced.
  • This fraction is, at least, larger than 0.01 %, preferably larger than 0.1 %, more preferably larger than 1 %, the most preferably larger than 10 %.
  • Such extracellular organelle vesicles are produced having none, one, or several contacts with other extracellular organelle vesicles.
  • Such extracellular organelle vesicles are produced having none, one, or several contacts with cell components such as cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, plasma membrane.
  • said extracellular organelle vesicles are defined by at least one of the following features, and preferably all the following features:
  • said extracellular organelle vesicles have a luminal volume which is bilayer- bounded; have a surface-to-volume at least lower than 3 pm 4 , 2 pm 4 , 1.5 pm 4 , 1.2 pm 4 , 1 pm 4 , 0.85 pm 4 , 0.75 pm 4 , 0.66 pm 4 , 0.6 pm 4 , 0.5 pm 4 , 0.42 pm 4 , 0.38 pm -1 , 0.33 pm’ 1 , 0.28 pm’ 1 , 0.25 pm’ 1 , 0.2 pm’ 1 , 0.15 pm’ 1 .
  • said extracellular organelle vesicles are produced from organelles of the source cell encompassing endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosome, endosome, peroxisome, multivesicular bodies.
  • said extracellular organelle vesicles are not surrounded by the plasma membrane of the source cell from which they are produced; meaning that said extracellular organelle vesicles are in an extracellular medium.
  • This biochemical composition is a fraction of the total composition of the organelle from which the extracellular organelle vesicles are produced. This fraction is, at least, larger than 0.01 %, 0.1 %, 0.5 %, 1 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 90 %, 100 %.
  • said extracellular organelle vesicles have a volume at least larger than 5 pm 3 , 15 pm 3 , 30 pm 3 , 60 pm 3 , 100 pm 3 , 180 pm 3 , 260 pm 3 , 380 pm 3 , 520 pm 3 , 1000 pm 3 , 1500 pm 3 , 2000 pm 3 , 3000 pm 3 , 4500 pm 3 .
  • said extracellular organelle vesicles have a size at least larger than 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 18 pm, 20 pm in all spatial directions.
  • said extracellular organelle vesicles have none, one, or several contacts with other GEOVs.
  • extracellular organelle vesicles have none, one, or several contacts with cell components such as cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, peroxisomes, plasma membrane and other organelles.
  • cell components such as cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, peroxisomes, plasma membrane and other organelles.
  • Said extracellular organelles vesicles can be functional further characterized in that said GEOVs transport neutral species and ions (membrane transport protein activity that is ranging from at least 0.1 %, 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % of that of the membrane transport activity in a reference cell, e.g., the source cell); comprise protein activity that is ranging from at least 0.1 %, 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % of that of the membrane transport activity in a reference cell, e.g., the source cell.
  • membrane transport protein activity that is ranging from at least 0.1 %, 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %
  • the extracellular organelle vesicles can be stored, and later delivered for research purposes.
  • the extracellular vesicles are stable at a temperature of less than 4°C, or -20°C, or -80°C for at least 1, 2, 3, 6, or 12 hours; 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, or 4 weeks; 1, 2, 3, or 6 months; or 1, 2, 3, 4, or 5 years.
  • Said extracellular organelle vesicles can be produced by a first or second process, said first and second processes being described below.
  • Such processes control lysis parameter to minimize organelle damages to recover highly functional organelles vesicles.
  • the second process is a reproducible process of extraction where each cell will be subjected to the same stress, being thus compatible with industrial requirements : volume production, organelle viability and reproducibility.
  • the first process is able to generate, extract and recover stable and functional organelles from cells, i.e. to produce said extracellular organelle vesicles.
  • Extracellular organelle vesicles can be produced from any cells, according to a first process comprising at least the following steps: u) Contacting the cells during 0.5 to 30 minutes with an hypotonic aqueous medium with an osmolarity ranging from 0.1 to 100 mOsm/L (for swelling both cells and its intracellular bilayer-bounded organelles, disrupting cell both cytoskeleton and extracellular matrix, spherizing the cell); v) Applying a membrane tension on cells ranging from 10' 3 to 5 mN/m, during 10' 4 to 100 seconds (for lysis and the removal of cell plasma membranes); w) Recovery of extracellular organelle vesicles into the hypotonic aqueous medium.
  • a first process comprising at least the following steps: u) Contacting the cells during 0.5 to 30 minutes with an hypotonic aqueous medium with an osmolarity ranging from 0.1 to 100 mOsm/L (for swelling both cells and its intracellular bilayer-
  • the cells from which the extracellular organelle vesicles are obtained can be cultured on a support or in suspension in bulk in any appropriate medium well known from the art, or can be recovered from organoids, tissues, organs or organisms.
  • Any type of cells can be used to generate organelles according to the first process.
  • cells derived from Cos7, Huh, mammalian cells, human cells, tumor cells obtained from the market, laboratories and hospital patients.
  • step u) is performed using a hypotonic aqueous medium which is any aqueous solution with an osmolarity ranging from 0.1 to 100 mOsm/L, preferably from 1 to 50 mOsm/L, more preferably from 5 to 50 mOsm/L, the most preferably from 10 to 40 mOsm/L, during 0.5, 3, 5, 7, 10, 15, 20 to 30 minutes, derived from, for example, buffer solutions (Diluted DPBS), diluted cell culture medium (DMEM), ionic solutions, salt solutions (e.g. CaC or KCI solutions), diluted buffer, water, etc..., for the generation of stable and functional giant organelle vesicles.
  • buffer solutions Diluted DPBS
  • DMEM diluted cell culture medium
  • salt solutions e.g. CaC or KCI solutions
  • the cytoplasm osmolarity is around 300 mOsm/L, meaning that all kind of aqueous solutions with an osmolarity below about 100 mOsm/L and higher than 0.1 mOsm/L, can be used to generate giant organelle vesicles.
  • Such hypotonic aqueous medium enables to instantly apply to cells and its intracellular organelles an adequate non-destructive, fast and effective osmotic shock to generate spherical swollen cells and enlarged organelle vesicles. Otherwise, the swelling protocols are too long and not effective, leading to non-spherical compartments and protein degradation, which makes the production of GEOVs impossible.
  • the control of the swelling kinetics parameters can favor the production of giant extracellular organelle vesicles.
  • Step u) is a step of osmotic swelling to generate intracellular organelles having an increased surface-to-volume ratio compared to their original form.
  • aqueous media is eventually combined with chemicals, to increase the size of intracellular organelle vesicles (bilayer-bounded compartments) leading to the formation of enlarged vesicle of a size distribution never reported in the prior literature, called giant (intracellular) organelle vesicles (GIOVs) and, to reduce the plasma membrane lysis tension value.
  • GIOVs giant organelle vesicles
  • cytoskeleton disruptor such as nocodazole, navelbine, latrunculin A, latrunculin B, cytochalasin; kinesin, myosin and dinein motor inhibitors such as blebistatin, benzytoluen sulphonamide, butanediome monoxime
  • cytoskeleton disruptor such as nocodazole, navelbine, latrunculin A, latrunculin B, cytochalasin; kinesin, myosin and dinein motor inhibitors such as blebistatin, benzytoluen sulphonamide, butanediome monoxime
  • the hypotonic medium of the first process can also contain one or more molecules to modulate the surface-to-volume ratio distribution of giant extracellular organelle vesicles while preventing their degradation and the value of surface tensions to lyse the plasma membrane (e.g. protease inhibitors, molecular motors inhibitors, organelle-cytoskeleton contact inhibitors, cytoskeleton disruptors, detergents). Some added molecules (e.g.
  • ion channels modulator/blocker thaspsigargin, caffein, benzothiasepin; Extra-cellular matrix distruptor such as trypsin; Protein transport inhibitors; Protein signaling inhibitors such as xelospongin; Chemical detergents such as Triton-X-100, octylglucoside, DDM, carboxylic acids) allow the cells to swell faster, and therefore to decrease the surface-to-volume ratio of produced giant extracellular organelle vesicles more quickly. Finally, the cell swelling kinetics is crucial not only to decrease the input energy to lyse the cell plasma membrane of step v) but also to control the size of the GEOVs produced at the end of step w).
  • the hypotonic aqueous medium comprises one or more molecules chosen from the group consisting of: nocodazole, latrunculins, trypsin, misakinolides, mycalolides, aplyronides, vinblastine, rotenone, swinholides, jasplakinolides, vincristine, demecolcine, cytochalasins, colchicine, vinca-alcaloids, dihydropyridine, phenylalkylamine, benzothiazepine, gabapentinoids, blebistatin, benzytoluen sulphonamide, butanediome monoxime, thaspsigargin, xelospongin, Triton X-100, Tween, SDS, Brij, Octyl Glucoside, octyl thioglucoside, CHAPS, CHAPSO, magnesium, said molecules being added before step u), at step u), at step u), at step
  • cells prior to the generation of giant extracellular organelle vesicles, cells may be treated with molecules to prevent protein degradation, modulate biochemical reactions on organelles, etc... so that giant extracellular organelle vesicles can bear specific biochemical properties.
  • the metabolic conditions and the architecture of cells and its organelles can thus be further modified before step v), by chemicals and/or by modifying the expression level of proteins impacting the architecture of cells and/or their organelles and/or the metabolic conditions.
  • cells can be treated with chemicals or can be modified to overexpress or repress the level of some proteins that impact the architecture of the cell and its organelles - notably the surface-to-volume ratio and the relative positioning of the organelles with each other - and the metabolic conditions.
  • the surface-to-volume ratio of the future produced giant extracellular organelles vesicles can be modulated (1) by altering the expression levels of proteins impacting organelle shape and contact sites with other organelles, plasma membrane and cytoskeleton, (2) by treating cells with molecules that alter: both the cytoskeleton and molecular motors, organelle contacts sites (with other organelles, plasma membrane and cytoskeleton), molecules-transporting-proteins activity localized on plasma membranes and organelles, signaling protein activity (3) by altering cellular metabolic pathways impacting organelles number and shape, surface, (4) any treatment mediate change in organelle surface-to-volume ratio and inter-organelle contact.
  • overexpressing climp63 prior to the generation and recovery of giant extracellular organelles vesicles leads to larger giant extracellular organelles vesicles emanating from the endoplasmic reticulum with sizes larger than 30 pm.
  • overexpressing Mfn2 prior to the generation and recovery of giant extracellular organelles vesicles leads to larger giant extracellular organelles vesicles emanating from the mitochondria with surface-to- volume ratio smaller than 0.75 pm -1 .
  • Pre-treating cells with nocodazole (or Latrunculin A) between 1 and 90 min before swelling allows the formation of bigger giant organelles from the ER.
  • rapamycin between 12 to 24 hours before swelling formation allows to form bigger giant extracellular organelles vesicles from endosome, lysosome, autolysosome and multivesicular body.
  • Adding bafylomicin before swelling and extraction also allows to obtain more giant extracellular organelles vesicles coming from autophagosomes.
  • said first process further comprises, after step u) and before step v), a step u') comprising generating a back-and-forth motion of the hypotonic aqueous medium to displace cells at a speed ranging from about 0.01 m/s to 10 m/s during about 0.01 seconds to 10 minutes, to disrupt both cytoskeleton and extracellular matrix of cells.
  • This optional step u') applied to swollen cells of step u) thus allows to further apply a less important stretching membrane tension in step v) to lyse cells, open them completely and release GEOVs without lysing them. This is because the cytoskeleton gives the plasma membrane of the cell an additional resistance.
  • the average lysis tension needed in step v) is about ⁇ 7 mN/m (some cells lyse at under ⁇ 1 mN/m and other at ⁇ 10 mN/m, as shown in example), whereas for swollen cells and organelles from step u) subjected to such motion in step u'), the average lysis tension needed in step v) is about ⁇ 2 mN/m (A large majority of cells lyses at tension under 1 mN/m, as shown in example).
  • the first process allows to drastically decrease ( ⁇ 2 mN/m) the membrane tension to reached cell lysis, the removal the whole plasma membrane and the release of all or almost all GEOVs from a cell, without lysing them thanks to gentle lysis.
  • this step u') where a back-and-forth motion of the hypotonic aqueous medium is generated to displace the cells at a speed ranging from 0.01 m/s to 10 m/s during 0.01 seconds to 10 minutes promotes the disruption of both the cytoskeleton and the extracellular matrix of the cells, in order to reduce the plasma membrane lysis tension and provoke plasma membrane full opening following lysis.
  • step v) is performed to apply a stretching membrane tension (bilayer tension) on cells ranging from 10‘ 3 to 5 mN/m, preferably from 5.10‘ 3 to 4 mN/m, more preferably from 10‘ 2 to 2 mN/m, even more preferably from 10‘ 2 to 1 mN/m most preferably between 5.10' 2 mN/m to 0.75 mN/m, during 10‘ 4 to 100 seconds, so as to break cell plasma membranes rapidly while preserving the structure of giant (extracellular) organelle vesicles released from lysed cells in the hypotonic aqueous medium.
  • a stretching membrane tension bilayer tension
  • the first process allows to lower the stretching membrane tension to values less than about ⁇ 2 mN/m to extract and recover GEOVs into the medium. While when the usual stretching membrane tensions known from the state of the art for lysing cells are on average higher than 5 mN/m, all the organelles are fragmented into extracellular nano-vesicles which release their content into the medium and are therefore no longer manipulate unlike claimed GEOVs.
  • Step v) can be carried out by using: mechanical force (e.g. suction pressure, stretching, shearing or acoustic wave), chemical agents and/or detergents, electric field, or laser (light) excitation.
  • the mechanical force is applied with a micro-pipette (radius from about 0,5 to 20 pm) with a suction pressure (generally ranging from about 0.1 to 600 mbar and more preferably from about 5 to 200 mbar (where previous bulk techniques use pressure much larger than 1 bar) that generates the expected stretching membrane tension on cells and leads to the lyse of plasma membrane and giant extracellular organelle vesicles recovery (see example section).
  • acoustic field pressure waves
  • 0.1 to 100 seconds allows to generate the expected stretching membrane tension on cells and leading to the lyse of plasma membrane and giant extracellular organelle vesicles recovery (see example section).
  • chemicals working as detergents can be added in a control manner to create the expected stretching membrane tension on cells that leads to the lyse of plasma membrane and giant extracellular organelle vesicles recovery, such as carboxylic acid (with concentrations sufficient to generate plasma membrane lysis), Triton X, Tween, SDS, Brij, Octyl Glucoside, octyl thioglucoside, CHAPS, CHAPSO and others.
  • Using detergents modifies the properties of the giant organelles.
  • step v) where cells' plasma membrane tension is increased mechanically from cells of step u) or steps u) + u') leads to the lysis and full removal of their plasma membrane and the release in the hypotonic medium of stable and functional giant extracellular organelle vesicles (GEOVs), without lysing them.
  • GEOVs giant extracellular organelle vesicles
  • the strong osmotic shock allows generate giant intracellular vesicles and to specifically destabilize the plasma membrane of cells, which then only needs a small mechanical perturbation to fully break and release the giant extracellular organelle vesicles, which are not broken thanks to the first process we developed for the plasma membrane lysis and removal.
  • the giant extracellular organelle vesicles of the first process make it now possible to turn toward applications previously unthinkable for industrial players. In addition, contact between such giant organelle vesicles can be conserved. Another advantage is the rapid isolation of giant organelle vesicles easily imaged with a microscope (e.g. about 5 to 20 pm sized).
  • Such giant extracellular organelle vesicles are functional, can be labelled, have any protein wanted on them, and can be easily picked and manipulated.
  • the cells of step u) can comprise cells transfected with at least one organelle protein marker or receiving molecules reporting for organelles to facilitate organelle identification.
  • the cells are transfected with at least one organelle protein tagged with a fluorescent or chemical marker used before or after swelling.
  • KDEL marks the ER. lumen
  • Tom20 marks mitochondria
  • LC3B autophagosomes
  • Lampl marks lysosomes
  • Golgi7 marks the Golgi apparatus.
  • the extracellular organelle vesicles of the first process can be recovered with any type of protein of interest, in contact with different cellular components, or other giant extracellular organelle vesicles.
  • the extracellular organelle vesicles can be obtained according to a second process implementing a device for recovering organelle vesicles from cells, said device comprising an extraction unit configured to extract organelle vesicles from cells, said process comprising a step of extraction of organelle vesicles from cells with said extraction unit, said extraction unit comprising a microfluidic chip having one or more microchannels configured to receive a fluid flow containing said cells, said microchannels comprising one or more constriction portions configured to apply mechanical constraints on said cells while passing therethrough.
  • the second process makes it possible to produce large quantities of organelle vesicles to make purified and concentrated samples that reach the market.
  • the microfluidic extraction is ideal to reach flow rates of typically a million of large organelle vesicles extracted per hour continuously.
  • the high frequency extraction unit of the invention can be directly connected to a sorting machine.
  • the device for recovering organelle vesicles from cells comprises an extraction unit configured to extract organelle vesicles from cells, said extraction unit comprising a microfluidic chip having one or more microchannels configured to receive a fluid flow containing said cells, said microchannels comprising one or more constriction portions configured to apply mechanical constraints on said cells while passing therethrough.
  • Such a microfluidic chip makes it possible to extract organelle vesicles from cells at high frequency, being thus compatible with industrial requirements.
  • At least one of said constriction portions is connected to an upstream portion and/or a downstream portion of a same microchannel.
  • Said upstream portion preferably has a section which decreases, preferably monotonically, from an upstream part of the microchannel to said constriction portion.
  • Said downstream portion preferably has a section which increases, preferably monotonically, from said constriction portion to a downstream part of the microchannel.
  • said upstream portion is formed by: - at least one first wall member defining with a transverse plane an angle ranging from 91° to 179°, preferably from 99° to 170°, for instance 105° or 130°, and/or
  • At least one second wall member defining with said transverse plane an angle ranging from 90° to 170°, preferably from 90° to 160°, for instance 95° or 120°.
  • said downstream portion is formed by:
  • At least one first wall member defining with a transverse plane an angle ranging from 91° to 180°, preferably from 120° to 179°, for instance 150° or 170°, and/or
  • At least one second wall member defining with said transverse plane an angle ranging from 90° to 170°, preferably from 90° to 160°, for instance 95° or 120.
  • a constriction portion When a constriction portion is connected to both an upstream portion and a downstream portion, it is preferred that said upstream portion is longer than said downstream portion, in relation to the direction of fluid flow.
  • said constriction portion has:
  • - a width ranging from 4 pm to 15 pm, preferably from 7 pm to 13 pm, for instance 8, 9, 10, 11 or 12 pm, and/or
  • - a height ranging from 5 pm to 100 pm, preferably from 10 pm to 30 pm, for instance 25, 30, 35 or 40 pm, and/or
  • - a length ranging from 1 pm to 200 pm, preferably from 10 pm to 70 pm, preferably from 25 pm to 50 pm, for instance 20, 30, 40, 50 or 60 pm, and/or.
  • At least one of said constriction portions may have a polygonal cross-section, for instance a rectangular or trapezoidal cross-section.
  • At least one of said microchannels comprises several of said constriction portions, for example two or more constriction portions.
  • the constriction section is decreased from one to another constriction portion of a same microchannel, in relation to the fluid flow direction.
  • several of said microchannels comprises one or more of said constriction portions, respectively.
  • the device implemented in the second process further comprises a preparation unit configured to prepare said cells.
  • the process further comprises, before the extraction step, a step of preparing said cells with said preparation unit.
  • said preparation unit comprises a means to put the cells in contact with a hypotonic aqueous medium.
  • the cells are put in contact with said hypotonic aqueous medium.
  • hypotonic aqueous medium is as defined for the first process described above.
  • the device implemented in the second process further comprises a collecting unit configured to collect and/or to sort said organelle vesicles extracted from cells with said extraction unit.
  • the process comprises, after the extraction step, a step of collecting said organelle vesicles with said collecting unit.
  • said collecting unit is formed by a part of said microfluidic chip and/or includes an additional microfluidic chip.
  • the device implemented in the second process further comprises an observation unit configured to observe said organelle vesicles extracted from cells with said extraction unit.
  • the process further comprises, during and/or after the step of extraction, a step of observation of said organelle vesicles with said observation unit.
  • said observation unit includes a camera and/or a microscope, for example a confocal microscope.
  • the second process comprises an introduction of a fluid containing said cells within said microfluidic chip microchannels of said extraction unit with a flow rate ranging from 1 pb/min to 500 pb/min, preferably from 50 pL/min to 200 pL/min, for instance 75, 100, 125, 150 or 175 pb/min.
  • the extraction step in the extraction unit corresponds to step v) of said first process defined above
  • the preparation step of the cells in the preparation unit corresponds to step u) of said first process defined above
  • the collecting step in the collecting unit corresponds to step w) of said first process defined above.
  • the invention relates as a second object to a kit for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein with respect to at least one compound C, wherein it comprises:
  • the extracellular organelle vesicle, the membrane transport protein and compound C are as defined in the second embodiment of the method according to the first object of the present invention.
  • the kit can optionally comprise a biological modulator reporting for the membrane transport protein, said biological modulator being as defined in the method according to the first object of the present invention.
  • Cos7, HeLa, HEK and fibroblast cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS heat inactivated Fetal Bovine Serum
  • penicillin-streptomycin 1% penicillin-streptomycin.
  • cells were cultivated 48h in DMEM media at 37°C with 5% CO2. Cells were cultured left adhering in Matek dishes, pre-treated or not with adhesion agents.
  • Cells were transfected 24h with different plasmids fused with fluorescent protein markers (e.g., RFP for Red Fluorescent Protein marker or BFP for Blue Fluorescent Protein marker), so as to probe different organelles before the extracellular organelle vesicles production. These plasmids serve to express proteins reporting for different organelles. Kdel-RFP or Erox-BFP and Mito-BFP plasmids were used to identify the endoplasmic reticulum and the mitochondria respectively.
  • fluorescent protein markers e.g., RFP for Red Fluorescent Protein marker or BFP for Blue Fluorescent Protein marker
  • An initial HKM buffer was prepared with 50 mM Hepes (known as 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid), 120 mM potassium acetate, and 1 mM MgC (in Milli-Q water) at pH 7.4. Said initial HKM buffer has an osmolarity of 280 ⁇ 15 mOsm/L.
  • the initial HKM buffer was diluted with H2O, pH 7.4, at 37°C and 5% CO2 to reach an osmolarity of 20 mOsm/L and form a diluted HKM buffer (hypotonic aqueous medium).
  • the DMEM medium is removed and the cells are brought into contact with the diluted HKM buffer during 15 minutes.
  • a back-and-forth motion of the hypotonic aqueous medium is then generated to displace cells during 5 seconds.
  • the back-and-forth motion is performed on all the volume of the hypotonic aqueous medium 3 times in a row. This step allows to destabilize the cytoskeleton of the cell and to reduce its lysis tension. At that step, organelle vesicles are still within the cells (i.e. intracellular organelle vesicles).
  • a membrane tension is applied on cells so as to produce extracellular organelle vesicles without lysing them.
  • the extracellular organelle vesicles are released (or produced) thanks to the lysis and the removal of the plasma membrane.
  • the membrane tension is carried out by micro-manipulation of the cells under confocal microscope thanks to a micropipette aspiration technique. Micropipette radius is around 1 pm. Thanks to a slight aspiration, a bilayer tongue of the plasma membrane was sucked into the micropipette. The aspiration is then increased at an approximate rate of 10 mbar/min, causing a proportional increase in the bilayer surface tension.
  • a certain applied tension e.g. surface tension ⁇ 0,75mN/m
  • the plasma membrane ruptured thanks to a pore opening in the plasma membrane and the extracellular organelle vesicles are released (or extracted).
  • micropipette After extraction, one or more extracellular organelle vesicles are isolated using a micropipette.
  • the micropipettes are used to hold a vesicle during the following methods of the present invention.
  • an acquisition is launched to observe the initial size of the vesicle.
  • Extracellular organelle vesicles are recovered and observed under a confocal microscope. Extracellular organelle vesicles can be observed thanks to the different above-mentioned markers (RFP) with a LSM800 Zeiss confocal microscope.
  • Micropipettes were made from capillaries (1.0 OD x 0.58 ID x 150 Lmm 30- 0017 GC100-15b; Harvard Apparatus) with a micropipette puller (model P-2000; Sutter Instruments). Micromanipulation was performed with TransferMan 4r (Eppendorf). Pressure measurement unit (DP103) was provided by Validyne Engineering.
  • Example 1 method for examining the transporting activity of Ca 2+ pump as membrane transport protein of the Endoplasmic Reticulum
  • the extracellular organelle vesicle held by the micropipette is passed through an aqueous solution containing CaC (60 mOsm/L) and ATP (100 pM).
  • aqueous solution containing CaC (60 mOsm/L) and ATP (100 pM).
  • CaC 60 mOsm/L
  • ATP 100 pM
  • Contacting calcium ions with said extracellular organelle vesicle increases the osmolarity of the extravesicular aqueous medium. Due to the difference in internal osmolarity of the vesicle (20 mOsm/L) and the CaC solution (60 mOsm/L), the vesicle deflates in a few seconds linked to an external water flow. In the following minutes, a re-swelling process is gradually observed.
  • This innovative process allows to examine the transporting activity of the ion pump SERCA localized on Endoplasmic Reticulum
  • Figure 1 A represents an extracellular organelle vesicle of endoplasmic reticulum (ER) which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L) as an extravesicular aqueous medium.
  • ER endoplasmic reticulum
  • KDEL ER resident protein
  • the ER extracellular organelle vesicle exhibits both its volume and fluorescence signal (respectively noted Vi and Fi) at initial state.
  • Vi and Fi fluorescence signal
  • t Os
  • CaCh 60 mOsm/L
  • ATP 100
  • t 20s
  • the ER. extracellular organelle vesicle is deflated : it reached its minimum volume (Vii) and its associated maximum fluorescence signal (Fii).
  • Figure 1 B represents plot of the mean vesicle fluorescence signal (F/Fi) and relative volume variations of vesicle V/Vi (measured in brightfield) and the associated opposite ratio (Vi/V).
  • F/Fi and Vi/V are highly correlated and seem proportional.
  • the ER extracellular organelle vesicle held by the micropipette is passed through an aqueous solution containing sucrose (60 mOsm). Due to the difference in internal osmolarity of the ER extracellular organelle vesicle (20 mOsm/L) and the sucrose solution (60 mOsm/L), the ER extracellular organelle vesicle deflates in a few seconds linked to an external water flow. However, in this case no re-swelling process is visible as shown in figure 2. Sucrose is thus not transported inside the ER extracellular organelle vesicle as it was the case of calcium ions with SERCA. Sucrose can thus be used as a control or reference in the method of the present invention.
  • the percentage of relative volume variation during the re-swelling phase which is followed under the microscope can give the relative activity of an ion channel. All parameters observable during the method of the present invention allow to obtain information on the activity of membrane transport proteins present on organelle vesicles.
  • the relative volume changes, the kinetics of volume change, the initial slopes of change, the values of the ratio between the initial volume and the volume after deflation and before re-inflation and/or of the ratio between the volume after deflation and before re-inflation and the final volume after re-inflation are relevant parameters to examine the transporting activity of a membrane transport protein.
  • Example 1 demonstrates applicability of the method according to the first object of the present invention, first embodiment.
  • figures 1 and 2 clearly show the transporting activity of the SER.CA ion pump as membrane transporting protein with respect to Ca 2+ as compound C with f/ ii > Vf, re f/Vii,ref, and f/ i > Vf,ref/Vi,ref, where sucrose is used instead of Ca 2+ as the reference compound.
  • Exemple 2 method for diagnosing a disease affecting a subject, said disease being characterized by abnormal expression and/or abnormal activity of Ca 2+ pump as a membrane transport protein of the Endoplasmic Reticulum (first object of the present invention, third embodiment)
  • Figure 3 A represents an extracellular organelle vesicle of endoplasmic reticulum (ER) which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L) as an extravesicular aqueous medium.
  • ER endoplasmic reticulum
  • KDEL ER resident protein
  • the ER extracellular organelle vesicle exhibits both its volume and fluorescence signal (respectively noted Vi and Fi) at initial state.
  • Figure 3 B represents plot of the mean vesicle fluorescence signal (F/Fi).
  • F/Fi mean vesicle fluorescence signal
  • Example 3 other parameters that can be used in the methods of the present invention
  • Figure 4 represents an ER extracellular organelle vesicle which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L).
  • This ER extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), overexpressed by COS7 cells, before organelle vesicles extraction.
  • KDEL ER resident protein
  • At t 0s, CaC (60 mOsm/L) and ATP (100
  • the series of bright field images show that during deflation and re-inflation, the vesicle remains patched with the micropipette, which makes it possible to measure the current in whole cell patch clamp conformation generated by the activity of ion pumps of the ER. extracellular organelle vesicle.
  • the membrane transport activity such as ion pump activity can be recorded using several simultaneous reading parameters:
  • the extracellular organelle vesicles are caught with micropipettes that are commonly used in patchclamp experiments.
  • the patchclamp is an electrophysiology method that allows recording the activity of ion channels by recording currents.
  • the extracellular organelle vesicles remain patched on the micropipettes in a conformation identical to the so called "cell-attached conformation" during a typical patch clamp experiment. It is therefore possible to record the activity of the ion pump on extracellular organelle vesicles while simultaneously observing the variation in current (recorded in patch clamp) and size (recorded in patch clamp).
  • the resident fluorescent proteins of the extracellular organelle vesicle are not permeable to its membrane. Thus they cannot cross the membrane during the swelling process.
  • the fluorescent signal (related to KDEL-RFP or ERox-BFP) increases, because the volume concentration of fluorescent proteins is higher.
  • the concentration of fluorescent protein decreases again, and thus the fluorescence signal as well.
  • extracellular organelle vesicles are « patchable » with a micropipette during the deflating-reswelling process.
  • Figure 5 represents an ER extracellular organelle vesicle which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L).
  • This extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), overexpressed by COS7 cells, before organelle vesicles extraction.
  • KDEL ER resident protein
  • COS7 cells were incubated during lh with a fluorophore called "Fluo8-AM" to report for calcium volumic concentration in organelles thanks to green fluorescence emission.
  • fluorophores than "Fluo8-AM” such as “Fluo3-AM” or “Fura-2” fluorophores can also be used to follow directly Ca 2+ concentrations variation inside ER extracellular organelle vesicles. These fluorophores are known to emit fluorescence that is proportional to Ca 2+ volumic concentration in a compartment.
  • Membrane surface tension parameter can be another way of examining the activity of a membrane transport protein: when the vesicle swells, its volume increases, as well as the area of its membrane that delimits it. This results in an increase in the surface tension of the membrane of the extracellular organelle vesicle which can be calculated simply with a micropipette and with the laws of Laplace.
  • fluorescent molecules such as "Flipper-TR” can be used to visualize a variation in the surface tension of the extracellular organelle vesicle membrane linked to the activity of ion channels causing the re-swelling.
  • Example 4 method for screening of candidate compounds intended to modulate the activity of Ca 2+ pump as a membrane transport protein of the Endoplasmic Reticulum (first object of the present invention, second embodiment)
  • thapsigargin (10 pM) in DMSO, is added to the medium.
  • Thapsigargin is a known inhibitor of the SERCA pump.
  • the extracellular organelle vesicle, held by the micropipette is passed through an aqueous solution containing CaC (60 mOsm/L) and ATP (ImM).
  • the vesicle Due to the difference in internal osmolarity of the extracellular organelle vesicle (20 mOsm/L) and the CaC solution (60 mOsm/L), the vesicle deflates in a few seconds linked to an external water flow. Surprisingly, the reswelling process is decreased or not present compared to the same experiment performed on ER extracellular organelle vesicle not incubated with the thapsigargin.
  • Figure 6 A represents an ER extracellular organelle vesicle maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L).
  • This extracellular organelle vesicle has been isolated thanks to an ER resident protein, over-expressed by COS7 cells, before organelle vesicles extraction.
  • Figure 6 B represents plot of the mean extracellular organelle vesicle fluorescence signal ( F/Fi) where corresponding volume of the vesicles are shown. This plot shows that, in presence of thaspigargin, the re-swelling process is almost off.
  • Example 4 demonstrates applicability of the method according to the first object of the present invention, second embodiment.
  • figure 1 and 6 clearly show the inhibitory effect of thapsigargin on the transporting activity of the SERCA ion pump as membrane transporting protein with respect to Ca 2+ as compound C with f/ ii ⁇ Vf, re f/Vii,ref, and f/ i ⁇ Vf, re f/Vi,ref, where the reference ratio is obtained without the presence of thapsigargin.
  • Example 5 method for determining the specificity of the SERCA pump as a membrane transport protein with respect to an extracellular organelle vesicle of endoplasmic reticulum type or the residency of the SERCA pump as a membrane transport protein in an extracellular organelle vesicle of endoplasmic reticulum type (first object of the present invention, fourth embodiment)
  • an Endoplasmic Reticulum extracellular organelle vesicle and a mitochondria extracellular organelle vesicle are isolated simultaneously with micropipettes, and visualized.
  • ER and mitochondria extracellular organelle vesicles were both put in contact with CaC (30 mOsm/L) and ATP (100 pM) during one hour.
  • Figure 7 represents Endoplasmic Reticulum extracellular organelle vesicle and mitochondria extracellular organelle vesicle are maintained thanks two micropipettes into a HKM diluted buffer (30 mOsm/L). These extracellular organelle vesicles were isolated thanks to ER and mitochondria resident proteins, over-expressed by COS7 cells, before organelle vesicles extraction.
  • Example 6 method for evaluating the response of the SERCA pump as a membrane transport protein contained in a membrane of an extracellular organelle vesicle of endoplasmic reticulum type with respect to a response of said membrane transport protein contained in a nucleus cell component (first object of the present invention, fifth embodiment)
  • an Endoplasmic Reticulum extracellular organelle vesicle and a nucleus cell component are isolated together from a same cell with one micropipette, and visualized.
  • ER extracellular organelle vesicle and nucleus cell component were both put in contact with CaC (20 mOsm/L) and ATP (100 pM) during 5 minutes.
  • Figure 8 represents Endoplasmic Reticulum extracellular organelle vesicle and nucleus cell component are maintained thanks one micropipette into a HKM diluted buffer (30 mOsm/L).
  • the extracellular organelle vesicle and the nucleus cell component were respectively isolated thanks to ER and nucleus probes, overexpressed by COS7 cells, before organelle vesicle and nucleus extraction.
  • the SERCA pump has a better response when localized in the membrane of an ER organelle vesicle compared to the nucleus cell component.
  • Example 7 method for evaluating the response of the SERCA pump as a membrane transport protein contained in a membrane of an extracellular organelle vesicle of endoplasmic reticulum type with respect to a response of said membrane transport protein contained in a plasma membrane cell component (first object of the present invention, fifth embodiment)
  • an Endoplasmic Reticulum extracellular organelle vesicle and a plasma membrane cell component are isolated with micropipettes.
  • an Endoplasmic Reticulum extracellular vesicle and a plasma membrane cell component do not come from the same cell.
  • SERCA protein is over-expressed in cell 1 on its native organelle, the endoplasmic reticulum.
  • SERCA is relocalized by genetic construction on the plasma membrane cell component. This finally serves to isolate with micropipettes, one endoplasmic reticulum extracellular vesicle with SER.CA extracted from cell 1 and one plasma membrane cell component enriched with SER.CA deriving from cell 2.
  • ER extracellular vesicle and plasma membrane cell component were both put in contact with CaC (60 mOsm/L) and ATP (100 pM) during 5 minutes.
  • the method of the present invention it is possible to demonstrate that the method of the present invention has a much better response than a well- known method which implements relocalization of the SERCA pump into the plasma membrane by genetic construction.
  • Example 8 method for examining the transporting activity of K + ion channel TMEM175 as membrane transport protein of the lysosome and endolysosome and the efficacy of drug candidates to modulate the transporting activity of TMEM175.
  • extracellular endolysosome vesicles are charged with K + ions thanks to a bath sonicator and an extracellular solution (210 mOsm/L) containing 100 mM of KCI and 10 mM of Hepes, pH 7.2. Then, extracellular endolysosome vesicles are seeded on a glass plate for observation. Before starting acquisition, extravesicular aqueous solution is removed to be changed when starting acquisition.
  • the extracellular organelle vesicle held by the micropipette is passed through an extra-vesicular aqueous solution (200 mOsm/L) containing 1 mM of KCI and 200mM of sucrose.
  • an extra-vesicular aqueous solution 200 mOsm/L containing 1 mM of KCI and 200mM of sucrose.
  • Contacting very small amount of potassium ions with said extracellular organelle vesicle generates a concentration gradient of K + between the inside and the outside of the vesicles. Due to the difference in internal K + of the vesicle (100 mM) and K + in the external solution (1 mM), the vesicle deflates in a few seconds linked to an external potassium flow. In the following minutes, a re-swelling process is not observed.
  • This innovative process allows to examine the transporting activity of the ion channel TMEM175 localized on lysosomes or endolysosomes vesicles in absence and/or in presence of an antagonist (4-AP) and/or agonist (DCPIB).
  • the methods of the present invention can be combined with a patch clamp measurement on extracellular organelle vesicles.
  • the methods of the present invention concern ion channels / ion pumps / transporters that are released and localized on extracted organelle vesicles : Endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, endolysosome, autophagosome, autolysosomes, endosomes, multivesicular bodies or a complex of 2 or more organelles in contact.
  • the methods of the present invention can be used in the pharmaceutical market as :
  • Figure 9 A) represents an extracellular organelle vesicle 1 provided in an extravesicular aqueous medium 2, said extracellular organelle vesicle 1 comprising a membrane 3 surrounding an intravesicular aqueous medium 4.
  • a membrane transport protein 5 is contained in said membrane 3.
  • Figure 9 B) represents the contacting step of said extracellular organelle vesicle 1 with a compound 6 (e.g. compound C as defined in the present invention) in the presence of a biomodulator 7 of the membrane transport protein 5.
  • the membrane transport protein 5 has a transporting activity with respect to said compound 6.

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Abstract

The present invention relates to several methods which implement morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and/or to study the biological behaviour of membrane transport proteins In particular, the5 invention relates to the monitoring of intracellular ion channels, ion pumps and transporters activity thanks to morphological changes of extracellular organelle vesicles.

Description

A METHOD IMPLEMENTING MORPHOLOGICAL CHANGES OF EXTRACELLULAR ORGANELLE VESICLES SO AS TO EXAMINE THE TRANSPORTING ACTIVITY OF MEMBRANE TRANSPORT PROTEINS AND OTHER RELATED METHODS
The present invention relates to several methods which implement morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and/or to study the biological behaviour of membrane transport proteins. In particular, the invention relates to the monitoring of intracellular ion channels, ion pumps and transporters activity thanks to morphological changes of extracellular organelle vesicles. More particularly, the present invention includes an in vitro or ex vivo method for examining the transporting activity and/or studying the biological behaviour of at least one membrane transport protein with respect to at least one specific compound, an in vitro or ex vivo method to determine the presence of at least one membrane transport protein in at least one extracellular organelle vesicle of a specific type, an in vivo or ex vivo method for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein, an in vitro or ex vivo method for determining abnormal expression and/or abnormal transporting activity of at least one membrane transport protein, and an in vitro or ex vivo method for evaluating the response of at least one membrane transport protein contained in a membrane of an extracellular organelle vesicle with respect to a response of said membrane transport protein contained in a cell component.
Transport membrane proteins such as ion channels represent an essential pharmacological target for drugs such as painkillers, anticonvulsants and neuromuscular blockers. The hit discovery rate of drugs for channels is severely impeded by several parameters: accessibility of the ion channel in a relevant membrane environment, and the cost and robustness of the current readout methods. In particular, most ion channels/pumps studied so far are localized at the plasma membrane. The membrane delineates the cell and limits access to intracellular ion channels/pumps. Currently, there is no means to easily access to and study intracellular ion channels/pumps. Moreover, heavy and costly methods are currently implemented by pharmaceuticals to measure the activity of ion fluxes across pumps/channels to develop drugs against these. The current gold standard approaches (only used so far for plasma membrane channels) require multiple upstream steps. Two methods are indeed used today for screening the activity of ion channels, localized at the plasma membrane: fluorescence monitoring, and patch clamp (electrophysiology). In the first method, specific fluorescence probes are used to detect overall alterations in intracellular ions, following the passage of the ion across the plasma membrane channels/pumps. This method is used on well plates to pre-screen drugs (before further studies in patch clamp) that modulate the activity of channel/pumps. This method does not allow to confidently assess the passage of ions at the organelle level. In the second method, ions fluxes are measured thanks to electrodes, to perform manual or automated patch clamp measurements. This approach is currently the gold standard for validating drug candidates. Much efforts have been developed for High Throughput Screening (HTS) patch clamp at the cell level. Because of the size of the electrodes and micropipettes used in patch clamp, large membranes (such as of the plasma membrane of cells) are required. The patch clamp method is not suitable for organelles because of their tiny size of organelles (in the few tens of nm) which, moreover, are currently not accessible.
Accordingly, there is still a need to measure robustly the activity of intracellular ion channels/pumps to develop more relevant and efficient drug targets. More generally, there is a need to examine the behaviour of all transport membrane proteins.
As a result of intensive research, the present inventors have found fast and reliable method for analysis of transport membrane protein activity. Said method allows for example the discovery of molecules that can modulate the activity of intracellular transport membrane proteins channeling passage of ions or solutes across membranes. Additionally, a lot of ion channels are crucial for our health, and, if some drug candidate alters their activity, it can lead to toxicity. Thus, the method of the present invention which allows the reading of ion channels activity can serve as efficacy tests but also for toxicity tests. Indeed, the method of the present invention is focused on quantifying the size/morphology change, and/or luminal fluorescence and/or surface membrane tension of specific extracellular organelle vesicles so as to examine the activity of a transport membrane protein with respect to a given molecule.
A first object of the present invention is an in vitro or ex vivo method for examining the transporting activity and/or studying the biological behaviour of at least one membrane transport protein with respect to at least one compound C, wherein said method comprises at least the following steps: i) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, ii) contacting said compound C with said extracellular organelle vesicle, and iii) maintaining said compound C and said extracellular organelle vesicle into contact, wherein said method further comprises:
- observing and/or measuring morphological change(s) of said extracellular organelle vesicle between step i) and step iii) and/or between step ii) and step iii), said morphological change(s) resulting in a change in at least one parameter, preferably selected from a volume V (in pm3), a membrane surface tension T (in N/m), and a luminal fluorescence value F (in arbitrary unit),
- comparing said morphological change(s) of said extracellular organelle vesicle with reference morphological change(s), wherein said membrane transport protein is contained in at least one of said membrane or a membrane of a reference extracellular organelle vesicle.
Thanks to the simple observation and/or the measurement of morphological change(s) of said extracellular organelle vesicle between step i) and step iii) and/or between step ii) and step iii), and the comparison of said morphological changes of said extracellular organelle vesicle with reference morphological changes, it is possible to study the biological behaviour and/or to examine the transporting activity of said membrane transport protein with respect to compound C.
In the present invention, compound C can also be defined as a molecule likely to be transported by a membrane transport protein.
Organelles used in the method of the present invention are biological material initially present in cells that are extracted from said cells in the form of vesicles. Such organelles are therefore outside of their hosting cell (hence the name "extracellular") in the form of vesicles before implementing the method of the present invention.
Consequently, the method of the present invention does not require the extraction or isolation of membrane transport proteins, which are localized in their original membrane and in their original organelle. The membrane transport proteins examined in the method of the present invention are only extracted from their hosting cell while maintaining their integrity.
The method of the invention is a method which avoids the denaturation, extraction and/or isolation of membrane transport proteins and which makes it possible to study them in their "native" environment, i.e. in their hosting organelle vesicle. This is totally different from methods of the prior art which implement reconstitution of membrane transport proteins in artificial models based on DIB ("droplet-interface bilayer"), SUV ("small unilamellar vesicle") and/or GUV ("giant unilamellar vesicle").
The extracellular organelle vesicles can be extracted from mammalian, plant and/or bacteria cells, and preferably extracted from mammalian cells.
Examples of such cells include COS-7 cells, HeLa cells, HEK cells, CHO cells, fibroblast cells, red blood cells, T-cells, neuroblastoma cells, stem cells, iPSC (induced Pluripotent Stem Cells) derived into cell types such iPSC derived neuronal stem cells, iPSC derived mesenchymal stem cells, iPSC derived monocytes stem cells, iPSC derived cardiomyocyte stem cells, iPSC derived microglia stem cells, iPSC derived myotubes stem cells, etc ...
Said cells can be obtained from the market, from laboratories and/or from hospitals, and/or recovered from organoids, biopsies, tissues, organs and/or organisms, from healthy or diseased human patient origin.
The morphological change(s) can occur between step i) and step iii), between step ii) and step iii), or both between step i) and step iii) and between step ii) and step iii). In the method of the present invention, the comparison is generally made between corresponding (or same) features. In other words, a morphological change occurring between step i) and step iii) is compared to a reference morphological change occurring between step i) and step iii), etc...
Observation of morphological change(s) of said extracellular organelle vesicle can be carried out by means such as confocal microscopy.
In the present invention, fluorescence variation can be analysed simultaneously with high throughput screening machine for imaging such as Molecular Devices, from Hamatsu's et al.
Morphological changes can be selected from deflation and inflation (also called re-swelling).
Said morphological change(s) of said extracellular organelle vesicle between step i) and step iii) and/or between step ii) and step iii) can be compared with morphological changes occurring with a same method where one parameter has been modified such as with a different compound instead of compound C, with addition of a new compound such as a drug candidate, with an extracellular organelle vesicle of the same type but from a different cell, with an extracellular organelle vesicle of a different type, for example from a same or different cell, or with one or more cellular components, for example from a same or different cell.
Said membrane transport protein is contained in at least one of said membrane or a membrane of a reference extracellular organelle vesicle. In other terms, said membrane transport protein is contained in said membrane, in a membrane of a reference extracellular organelle vesicle, or both in said membrane and in a membrane of a reference extracellular organelle vesicle.
Said morphological change(s) results in a change in at least one parameter, preferably selected from a volume V (in pm3), a membrane surface tension T (in N/m), and a luminal fluorescence value F (in arbitrary unit).
The volume V relates to the volume of extracellular organelle vesicle, the membrane surface tension T relates to the membrane surface tension T of the membrane of the extracellular organelle vesicle and the luminal fluorescence value F relates to the luminal fluorescence value F of the intravesicular aqueous medium.
The luminal fluorescence value F can be obtained thanks to grafting or labelling a resident protein of the extracellular organelle vesicle (as explained later in the processes to obtain said extracellular organelle vesicles). The luminal fluorescence is by definition fluorescence of lumen. The luminal fluorescence can be obtained by labelling luminal proteins or luminal metabolites. The luminal fluorescence has therefore to be distinguished from fluorophore reporting the concentration of compound C which can be obtained by using molecules that complex with compound C and emit fluorescence when interacting with compound C (Ex : FURA2, Fluo4, Fluo8). Such fluorescence of compound C does not corresponds to a parameter characterizing the extracellular organelle vesicle, and even less to a parameter characterizing the morphological change of said extracellular organelle vesicle. The luminal fluorescence, contrary to the fluorescence of compound C, is independent from the concentration of compound C. The labelling of compound C (with at least one fluorophore molecule reporting for the compound C) can be performed as an additional tool as explained in example 3.
The method can be carried with one or more extracellular organelle vesicles. Step i) can implement several extracellular organelle vesicles of the same type or several extracellular organelle vesicles of different types, such as for example of at least two different types.
In step i), said extravesicular aqueous medium and said intravesicular aqueous medium are preferably in a state of equilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have identical or almost identical osmolarities (in mOsm/L).
In the present invention, the term "almost identical" means that a difference of at most 20%, and preferably at most 10%, can be found between both osmolarities.
In one preferred embodiment, the contacting step ii) leads to a state of desequilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have different osmolarities (in mOsm/L).
In one preferred embodiment, the contact maintaining step iii) leads to a return to equilibrium, in particular said extravesicular aqueous medium and said intravesicular aqueous medium have identical or almost identical osmolarities (in mOsm/L).
Step iii) is preferably carried out during at least 10 seconds.
First embodiment
In a first embodiment, the method according to the first object of the present invention comprises at least the following steps: i) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, and said extracellular organelle vesicle being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii) contacting said compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm3), a membrane surface tension Tn, and a luminal fluorescence value Fii (in arbitrary unit), iii) maintaining said compound C and said extracellular organelle vesicle into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a final volume f (in pm3), a final membrane surface tension Tf, and a final luminal fluorescence value Ff (in arbitrary unit), iv) calculating at least one ratio selected from Vf/Vii, Tf/Tn, Ff/Fn, f/ i, Tf/Ti, and Ff/Fi, v) comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff,ref/Fil,ref, Vf, ref/ Vi, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref, SO 8S tO evaluate the transporting activity and/or the behaviour of said membrane transport protein with respect to said compound C, wherein the corresponding reference ratio is obtained by applying said method [i.e. steps i) to iv)] with a reference compound instead of said compound C, said membrane transport protein not having a transporting activity with respect to said reference compound or said membrane transport protein having a known transporting activity with respect to said reference compound.
The calculation of a ratio as defined in step iv) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle. However, other means can be used.
As mentioned above, in the method of the present invention, the comparison is generally made between corresponding (or same) features. Thus, in this embodiment, corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf,ref/Vii,ref, or Vf/Vi is compared with Vf,ref/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf,ref/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff,ref/Fi,ref.
In one preferred embodiment, transporting activity of said membrane transport protein with respect to said compound C is found when at least one of the following equation is verified:
- Vf/Vil > Vf,ref/Vil,ref,
- Tf/Til > Tf,ref/Til,ref,
- Ff/Fil < Ff, ref/ Fi 1 , ref ,
- Vf/Vi > Vf,ref/Vi,ref,
- Tf/Ti > Tf,ref/Ti,ref,
- Ff/Fi < Ff,ref/Fii,ref, and when the corresponding reference ratio is obtained by applying said method with a reference compound instead of said compound C, said membrane transport protein not having a transporting activity with respect to said reference compound.
A rate of transporting activity (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method with a first reference compound instead of said compound C, said membrane transport protein not having a transporting activity with respect to said first reference compound and the second corresponding reference ratio is obtained by applying said method with a second reference compound instead of said compound C, said membrane transport protein having a known good transporting activity with respect to said second reference compound.
The measuring in step ii) [respectively the measuring in step Hi)] can be dynamic or static. In other words, the measurement in step ii) [respectively in step Hi)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
Depending on the ratio to be calculated, the measuring in step ii) is not required.
Second embodiment
In a second embodiment, the method according to the first object of the present invention is a method for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein with respect to at least one compound C, wherein it comprises at least the following steps: a) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, and said extracellular organelle vesicle being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), b) contacting said compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit) of said extracellular organelle vesicle, wherein said membrane transport protein has a transporting activity with respect to said compound C, c) contacting at least one candidate compound with said extracellular organelle vesicle, wherein step c) is carried out before step b) or after step b), and preferably before step b), d) maintaining said compound C, said candidate compound, and said extracellular organelle vesicle into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a final volume Vf (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit), e) calculating at least one ratio selected from Vf/Vii, Tf/Tii, Ff/Fii, Vf/Vi, Tf/Ti, and Ff/Fi, f) comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf, ref/ Vi, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the transporting activity and/or the behaviour of said membrane transport protein with respect to compound C in the presence of said candidate compound, wherein the corresponding reference ratio is obtained by applying said method without the presence of said candidate compound [i.e. steps a), b) d), and e)] or by applying said method [i.e. steps a) to e)] with a reference candidate compound instead of said candidate compound, said reference candidate compound having a known modulator effect on the transporting activity of said membrane transport protein with respect to said compound C.
The calculation of a ratio as defined in step e) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle. However, other means can be used.
As mentioned above, in the method of the present invention, the comparison is generally made between corresponding (or same) features. Thus, in this embodiment, corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf,ref/Vii,ref, or Vf/Vi is compared with Vf,ref/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf,ref/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff,ref/Fi,ref.
In one preferred embodiment, inhibitory effect of said candidate compound is found when at least one of the following equation is verified:
- Vf/Vil < Vf,ref/Vil,ref,
- Tf/Til < Tf,ref/Til,ref,
- Ff/Fil > Ff, ref/ Fi 1 , ref ,
- Vf/Vi < Vf,ref/Vi,ref, - Tf/Ti < Tf,ref/Ti,ref,
- Ff/Fi > Ff,ref/Fi,ref.
A rate of inhibitory activity (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method without the presence of said candidate compound and the second corresponding reference ratio is obtained by applying said method with a reference candidate compound instead of said candidate compound, said reference candidate compound having a known good inhibitory effect on the transporting activity of said membrane transport protein with respect to said compound C.
The measuring in step b) [respectively the measuring in step d)] can be dynamic or static. In other words, the measurement in step b) [respectively in step d)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
Depending on the ratio to be calculated, the measuring in step b) is not required.
The contacting step c) is preferably carried out with a solution (e.g. aqueous solution) comprising said candidate compound at a molar concentration ranging from 0.001 pM to 100 mM, and preferably ranging from 0.1 nM to 100 pM.
The contacting step c) is preferably performed during at least 5 seconds, and more preferably during at least 30 seconds.
The method according to the second embodiment can be carried out with extracellular organelle vesicles of different types so as to evaluate toxicity of said candidate compound with respect to some of said different types of extracellular organelle vesicles.
For example, it can be useful to implement such method to identify candidate compounds that are good inhibitors of the transporting activity of a membrane transport protein from a specific organelle but which do not have inhibitory activity of said membrane transport protein from other types of organelles. This ensures selectivity of the inhibitor.
Third embodiment
In a third embodiment, the method according to the first object of the present invention is a method for determining abnormal expression and/or abnormal transporting activity of at least one membrane transport protein with respect to at least one compound C, comprising at least the following steps:
A) providing in an extravesicular aqueous medium at least one extracellular organelle vesicle from genetically-modified cells or from cells of a patient, said extracellular organelle vesicle comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, and said extracellular organelle vesicle being described by at least one parameter selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit),
B) contacting said compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit),
C) maintaining said compound C and said extracellular organelle vesicle into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit),
D) calculating at least one ratio selected from f/ ii, Tf/Tii, Ff/Fii, f/ i, Tf/Ti, and Ff/Fi,
E) comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf, ref/ i, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the abnormal expression and/or abnormal transporting activity of said said membrane transport protein with respect to said compound C, wherein the corresponding reference ratio is obtained by applying said method [i.e. steps A) to D)] to a reference extracellular organelle vesicle from cells of an healthy patient or from non-genetically-modified cells, and wherein said membrane transport protein has transporting activity with respect to said compound C in said cells of a healthy patient or in said non-genetically-modified cells.
In this embodiment, the method can be implemented to diagnose (/n vivo and/or ex vivo diagnostic) a disease affecting said patient, said disease being characterized by abnormal expression and/or abnormal transporting activity of said at least one membrane transport protein with respect to said compound C. The calculation of a ratio as defined in step D) is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle. However, other means can be used.
As mentioned above, in the method of the present invention, the comparison is generally made between corresponding (or same) features. Thus, in this embodiment, corresponding ratios are compared. More particularly, Vf/Vii is compared with Vf,ref/Vii,ref, or Vf/Vi is compared with Vf,ref/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf,ref/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff,ref/Fi,ref.
In one preferred embodiment, overexpression of said membrane transport protein is found when at least one of the following equation is verified:
- Vf/Vil > Vf,ref/Vil,ref,
- Tf/Til > Tf,ref/Til,ref,
- Ff/Fil < Ff, ref/ Fi 1 , ref ,
- Vf/Vi > Vf,ref/Vi,ref,
- Tf/Ti > Tf,ref/Ti,ref,
- Ff/Fi < Ff,ref/Fi,ref.
A rate of altered transport protein activity of a patient from a disease related to said membrane transport protein (in %) can be calculated by comparing the ratio with at least two corresponding reference ratios, wherein the first corresponding reference ratio is obtained by applying said method to a first reference extracellular organelle vesicle from cells of an healthy patient and the second corresponding reference ratio is obtained by applying said method to a second reference extracellular organelle vesicle from cells of an sick patient.
The measuring in step B) [respectively the measuring in step C)] can be dynamic or static. In other words, the measurement in step B) [respectively in step C)] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
Depending on the ratio to be calculated, the measuring in step B) is not required.
Fourth embodiment In a fourth embodiment, the method of the first object of the present invention is a method to determine the presence of at least one membrane transport protein in at least one extracellular organelle vesicle of type 1, wherein said method comprises at least the following steps: i') providing in an extravesicular aqueous medium at least one extracellular organelle vesicle of type 1, said extracellular organelle vesicle of type 1 comprising a membrane surrounding an intravesicular aqueous medium, said extracellular organelle vesicle of type 1 being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii') contacting at least one compound C with said extracellular organelle vesicle of type 1, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle of type 1 selected from a volume Vn (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit), wherein said at least one membrane transport protein has a transporting activity with respect to said compound C,
Hi') maintaining said compound C and said extracellular organelle vesicle of type 1 into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle of type 1 selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit), iv') calculating at least one ratio selected from f/ ii, Tf/Tii, Ff/Fu, f/ i, Tf/Ti, and Ff/Fi, v') comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf, ref/ i, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the presence of said membrane transport protein in said at least one extracellular organelle vesicle of type 1, wherein the corresponding reference ratio is obtained:
* by applying said method [i.e. steps i') to iv')] with an extracellular organelle vesicle of a reference type instead of said extracellular organelle vesicle of type 1, said extracellular organelle vesicle of a reference type comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein, or * by further providing in said extravesicular aqueous medium of said step i') an extracellular organelle vesicle of a reference type in addition to said extracellular organelle vesicle of type 1, said extracellular organelle vesicle of a reference type comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein.
The calculation of a ratio as defined in step iv') is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a reference extracellular organelle vesicle. However, other means can be used.
As mentioned above, in the method of the present invention, the comparison is generally made between corresponding (or same) features. Thus, in this embodiment, corresponding ratios are compared. More particularly, f/ ii is compared with Vf,ref/Vii,ref, or f/ i is compared with Vf,ref/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf,ref/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff,ref/Fi,ref.
The measuring in step ii') [respectively the measuring in step Hi')] can be dynamic or static. In other words, the measurement in step ii') [respectively in step Hi')] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
Depending on the ratio to be calculated, the measuring in step ii') is not required.
In the method as defined above, the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 can come from a same cell or a different cell.
In the method as defined above, the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 can be present (or comprised) in said extravesicular aqueous medium of step i '), with physical contact or without physical contact.
More particularly, when the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 come from a same cell, they are in physical contact in said extravesicular aqueous medium of step i'). When the extracellular organelle vesicle of a reference type and the extracellular organelle vesicle of type 1 come from a different cell, they may be or not be in physical contact in said extravesicular aqueous medium of step i'). The method according to the fourth embodiment can be used to determine the activity of a membrane transport protein which is present in two different types of organelles but without the same density and/or without the same membrane environment.
Fifth embodiment
In a fifth embodiment, the method of the first object of the present invention is a method to evaluate the response of at least one membrane transport protein contained in a membrane of an extracellular organelle vesicle with respect to a response of said membrane transport protein contained in a cell component, wherein said method comprises at least the following steps:
I') providing in an extravesicular aqueous medium at least said extracellular organelle vesicle, said extracellular organelle vesicle comprising said membrane surrounding an intravesicular aqueous medium, said extracellular organelle vesicle being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit),
II') contacting at least one compound C with said extracellular organelle vesicle, and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit), wherein said at least one membrane transport protein has a transporting activity with respect to said compound C,
III') maintaining said compound C and said extracellular organelle vesicle into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit),
IV') calculating at least one ratio selected from Vf/Vii, Tf/Tii, Ff/Fu, Vf/Vi, Tf/Ti, and Ff/Fi,
V') comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff,ref/Fil,ref, Vf, ref/ Vi, ref, Tf,ref/Ti,ref, a nd Ff,ref/Fi,ref, wherein the corresponding reference ratio is obtained:
* by applying said method [i.e. steps I') to IV')] with a cell component instead of said extracellular organelle vesicle, or * by further providing in said extravesicular aqueous medium of said step I') a cell component in addition to said extracellular organelle vesicle.
The calculation of a ratio as defined in step IV') is one of the preferred means so as to compare morphological change(s) of said extracellular organelle vesicle with morphological change(s) of a cellular component. However, other means can be used.
As mentioned above, in the method of the present invention, the comparison is generally made between corresponding (or same) features. Thus, in this embodiment, corresponding ratios are compared. More particularly, f/ ii is compared with Vf,ref/Vii,ref, or f/ i is compared with Vf,ref/Vi,ref, or Tf/Tu is compared with Tf,ref/Tii,ref, or Tf/Ti is compared with Tf,ref/Ti,ref, or Ff/Fii is compared with Ff,ref/Fii,ref, or Ff/Fi is compared with Ff,ref/Fi,ref.
The measuring in step II') [respectively the measuring in step III')] can be dynamic or static. In other words, the measurement in step II') [respectively in step III')] can be performed once after the contacting step (static) [respectively after the contact maintaining step] or several times after the contacting step (dynamic) [respectively after the contact maintaining step].
Depending on the ratio to be calculated, the measuring in step III') is not required.
In the method as defined above, the cell component and the extracellular organelle vesicle can come from a same cell or a different cell.
In the method as defined above, the cell component and the extracellular organelle vesicle can be present (or comprised) in said extravesicular aqueous medium of step I'), with physical contact or without physical contact.
More particularly, when the cell component and the extracellular organelle vesicle come from a same cell, they are in physical contact in said extravesicular aqueous medium of step I'). When the cell component and the extracellular organelle vesicle come from a different cell, they may be or not be in physical contact in said extravesicular aqueous medium of step I').
The method according to the fourth embodiment can be used to determine the activity of a membrane transport protein which is present in cell components or to demonstrate the efficacy of implementing extracellular organelle vesicle(s) instead of cell components which are used in methods of the prior art. In one embodiment, the extracellular organelle vesicle in the method according to the first, second, third and fifth embodiments of the first object of the present invention is selected from organelles of endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, and multivesicular bodies, preferably is an extracellular organelle vesicle of Golgi apparatus, endoplasmic reticulum, mitochondria, lysosome, or endolysosome, and more preferably of endoplasmic reticulum.
In one embodiment, the extracellular organelle vesicle of type 1 (respectively the extracellular organelle vesicle of reference type) in the method according to the fourth embodiment of the first object of the present invention is selected from organelles of endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, and multivesicular bodies.
In one embodiment, the method according to the fourth embodiment of the first object of the present invention is implemented with several types of extracellular organelle vesicles, preferably from a same type of cell, so as to screen which type(s) of organelle vesicles has said at least one membrane transport protein as a resident protein, said several types of extracellular organelle vesicles being preferably selected from the following types: endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, multivesicular bodies.
As an example, the extracellular organelle vesicle of type 1 can be a extracellular mitochondria vesicle; and the extracellular organelle vesicle of reference type can be a extracellular endoplasmic reticulum vesicle.
In one embodiment of the method according to the fifth embodiment of the first object of the present invention, the cell component is selected from cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, and plasma membrane, and preferably selected from cell nucleus and plasma membrane.
In one embodiment of the method according to the fifth embodiment of the first object of the present invention, the cell component can be genetically enriched with said membrane transport protein. In other words, the membrane transport protein is overexpressedin said cell component. In the method according to the first object of the present invention (including first, second, third, fourth and fifth embodiments), the membrane transport protein is preferably an ion channel, an ion pump, or a transporter, and more preferably an ion channel or an ion pump.
Examples of ion pumps include Lysosomal proton pumps, Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps, Endoplasmic reticulum Ca2+ ATPase (ERCA) pumps, P-type ATPase pumps, Na+/K+ ATPase pumps, Ca2+/Mn2+ ATfPase pumps, Proton ATPase pumps, V-ATPase, Sodium/potassium-ATPase (Na+/K+-ATPase), H+ ATPase, P-type ATPase, Mg2+ ATPase, Ca2+ ATPase, ATP13A2/PARK9, preferably Lysosomal proton pumps, Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps, Endoplasmic reticulum Ca2+ ATPase (ERCA) pumps, Na+/K+ ATPase pumps, Ca2+/Mn2+ ATPase pumps, V-ATPase, H+ ATPase, P-type ATPase, Mg2+ ATPase, Ca2+ ATPase, and more preferably Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps.
Examples of ion channels include Inositol trisphosphate (IP3) receptors, Ryanodine receptors (RyRs), Lysosomal anion channel CLC-7, TMEM175 H+ channel, TMEM Channels, Transient receptor potential mucolipin 1 (TRPML1), Transient receptor potential mucolipin 2 (TRPML2), Transient receptor potential mucolipin 3 (TRPML3), Chloride channels CliC, Anion-selective channel 1 (ASCL1), Acid-sensing ion channels (ASICs), G-protein-gated acid-sensing ion channel (ASIC-like, GAC), Mitochondrial ATP-sensitive potassium (mKATP) channels, Mitochondrial voltagedependent anion-selective channel (VDAC), Calcium release-activated calcium (CRAC) channels, Store-operated calcium (SOC) channels, Inwardly rectifying potassium (Kir) channels, ATP-sensitive potassium (KATP) channels, Inwardly rectifying cation (IRC) channels, Voltage-gated potassium (Kv) channels, Transient receptor potential canonical (TRPC) channels, Transient receptor potential melastatin (TRPM) channels, Transient receptor potential vanilloid (TRPV) channels, Transient receptor potential mucolipin (TRPML) channels, Anion-selective channel 1 (ASCL1), G-protein-gated acid-sensing ion channel (ASIC-like, GAC), Voltage-gated potassium channel (Kv channel), Voltage-gated calcium channel (CaV channel), Ligand-gated ion channel (LGIC), Calcium-activated potassium channel (BK channel), Calcium- activated chloride channel (CaCC), Inward-rectifier potassium channel (Kir channel), Two-pore domain potassium channel (K2P channel), Transient receptor potential channel (TRP channel), Polycystic kidney disease 2-like 1 channel (PKD2L1 channel), Acid-sensing ion channel (ASIC), GABA receptor, NMDA receptor, AMPA receptor, Kainate receptor, Nicotinic acetylcholine receptor, Muscarinic acetylcholine receptor, Adenosine receptor, Serotonin receptor, Dopamine receptor, and preferably Inositol trisphosphate (IP3) receptors, Ryanodine receptors (RyRs), Lysosomal calcium channel TRPML1, Lysosomal anion channel CLC-7, TMEM175 H+ channel, TMEM Channels, Chloride channels CliC, Acid-sensing ion channels (ASICs), Mitochondrial ATP-sensitive potassium (mKATP) channels, Mitochondrial voltage-dependent anionselective channel (VDAC), Calcium release-activated calcium (CRAC) channels, NMDA receptor.
Examples of transporters include Lysosomal amino acid transporter 1 (LAT1), Lysosomal amino acid transporter 2 (LAT2), Cationic amino acid transporter 3 (CAT3), Lysosomal chloride transporter CLCN7, Lysosomal cystine transporter cystinosin, Lysosomal iron transporter NRAMP2, Lysosomal cholesterol transporter NPC1, Lysosomal sphingolipid transporter NPC2, Lysosomal glucose transporter GLUT5, Lysosomal glucose transporter GLUT7, Lysosomal fructose transporter
GLUT5, Lysosomal mannose transporter GLUT9, Lysosomal phospholipid transporter LIMP-2, Lysosomal nucleotide transporter CNT2, Lysosomal nucleotide transporter CNT3, Lysosomal nucleotide transporter SLC17A9, Lysosomal fatty acid transporter CD36, Lysosomal fatty acid transporter LIMP-2, Lysosomal folate transporter FOLR1, Lysosomal vitamin C transporter SVCT2, Lysosomal zinc transporter ZIP4, Lysosomal copper transporter CTR1, Lysosomal glutathione transporter ABCG2, Lysosomal lipid transporter MLN64, Lysosomal drug transporter ABCB1, Lysosomal drug transporter ABCG2, Lysosomal drug transporter SLC22A16, Lysosomal drug transporter SLC22A17, Sodium-dependent phosphate transporter 2b (NaPi2b), Proton-coupled folate transporter (PCFT), Zinc transporter 3 (ZnT3), Copper transporter 1 (CTR1), Na+/HCO3' cotransporter 3 (NBC3), Na+/dicarboxylate cotransporter 1 (NaDCl), SLC26A3/dicarboxylate transporter, SLC26A6/sulfate transporter, SLC26A7/chloride transporter, Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides,
SLC22A5/organic cation transporter, SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter, SLC22All/organic anion transporter, Mitochondrial phosphate transporter (PiC),
Mitochondrial carnitine/acylcarnitine transporter (CACT), Mitochondrial malate transporter (DIC), Mitochondrial adenosine nucleotide transporter (ANT), Mitochondrial citrate transporter (CiC), Mitochondrial folate transporter (MFT), Sodium-dependent phosphate transporter 2b (NaPi2b), Proton-coupled folate transporter (PCFT), Zinc transporter 3 (ZnT3), Copper transporter 1 (CTR1), Na+/HCO3’ cotransporter 3 (NBC3), Na+/dicarboxylate cotransporter 1 (NaDCl), SLC26A3/dicarboxylate transporter, SLC26A6/sulfate transporter, SLC26A7/chloride transporter, Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides, SLC22A5/organic cation transporter, SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter,
SLC22All/organic anion transporter, Lysosomal amino acid transporter 1 (LAT1), Lysosomal amino acid transporter 2 (LAT2), Cationic amino acid transporter 3 (CAT3), Lysosomal chloride transporter CLCN7, Lysosomal cystine transporter cystinosin, Lysosomal iron transporter NRAMP2, Lysosomal cholesterol transporter NPC1, Lysosomal sphingolipid transporter NPC2, Lysosomal glucose transporter GLUT5, Lysosomal glucose transporter GLUT7, Lysosomal fructose transporter
GLUT5, Lysosomal mannose transporter GLUT9, Lysosomal phospholipid transporter LIMP-2, Lysosomal nucleotide transporter CNT2, Lysosomal nucleotide transporter CNT3, Lysosomal nucleotide transporter SLC17A9, Lysosomal fatty acid transporter CD36, Lysosomal fatty acid transporter LIMP-2, Lysosomal folate transporter FOLR.1, Lysosomal vitamin C transporter SVCT2, Lysosomal zinc transporter ZIP4, Lysosomal copper transporter CTR.1, Lysosomal glutathione transporter ABCG2, Lysosomal lipid transporter MLN64, Lysosomal drug transporter ABCB1, Lysosomal drug transporter ABCG2, Lysosomal drug transporter SLC22A16, Lysosomal drug transporter SLC22A17, Sodium-dependent phosphate transporter 2b (NaPi2b), Proton-coupled folate transporter (PCFT), Zinc transporter 3 (ZnT3), Copper transporter 1 (CTR.1), Na+/HCO3' cotransporter 3 (NBC3), Na+/dicarboxylate cotransporter 1 (NaDCl), SLC26A3/dicarboxylate transporter, SLC26A6/sulfate transporter, SLC26A7/chloride transporter, Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides,
SLC22A5/organic cation transporter, SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter, SLC22All/organic anion transporter, Mitochondrial phosphate transporter (PiC),
Mitochondrial carnitine/acylcarnitine transporter (CACT), Mitochondrial malate transporter (DIC), Mitochondrial adenosine nucleotide transporter (ANT), Mitochondrial citrate transporter (CiC), Mitochondrial folate transporter (MFT), Sodium-dependent phosphate transporter 2b (NaPi2b), Proton-coupled folate transporter (PCFT), Zinc transporter 3 (ZnT3), Copper transporter 1 (CTR1), Na+/HCO3' cotransporter 3 (NBC3), Na+/dicarboxylate cotransporter 1 (NaDCl), SLC26A3/dicarboxylate transporter, SLC26A6/sulfate transporter, SLC26A7/chloride transporter, Sodium-dependent vitamin C transporter 1 (SVCT1), Sodium-dependent vitamin C transporter 2 (SVCT2), SLC29A3/transporter for nucleosides, SLC22A5/organic cation transporter, SLC22A7/organic anion transporter, SLC22A8/organic anion transporter, SLC22A9/organic anion transporter, SLC22All/organic anion transporter, Glutamate transporter, GABA transporter, Glycine transporter, Glucose transporter, Sodium/glucose cotransporter (SGLT), Proton-coupled oligopeptide transporter (PepT), Organic anion transporter (OAT), Organic cation transporter (OCT), ATP-binding cassette transporter (ABC transporter), Sodium/bicarbonate cotransporter (NBC), Sodium/hydrogen carbonate cotransporter (NBCe), Sodium-dependent phosphate transporter (NaPi), Glutamate transporter-like protein 3 (EAAT3), Glucose transporter 3 (GLUT3), SLClAl/sodium- dependent neutral amino acid transporter, SLC22A2/organic cation transporter, SLC22A8/organic anion transporter, SLC22A12/organic anion transporter, SLC22A14/organic anion transporter, SLC22A18/organic cation transporter, SLC22A20/organic cation transporter, Sec61 translocon complex.
Most preferred examples are SER.CA ion pumps of endoplasmic reticulum and TMEM and TPC lysosomal channels.
In the method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the compound C is preferably selected from ions, cellular metabolites, pyruvate, amino acids, fatty acids, fatty acids CoA, malate, citrate, isocitrate, and vitamins, and more preferably selected from ions.
Preferred examples of ions as compound C are Na+, K+, Ca2+, H+, HPC 2', HCCh' , Mg2+, Cl', Fe2+, Fe3+ and other ions generally present in a cell cytoplasm or in an organelle lumen, more preferably Na+, K+, Ca2+, H+, HPC 2', HCOs' , Mg2+, Cl', Fe2+, Fe3+, and even more preferably Ca2+.
Advantageously, in the method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the membrane transport protein is an ion channel or an ion pump and the compound C is selected from ions, and preferably selected from Na+, K+, Ca2+, H+, HPC 2', HCCh' , Mg2+, Cl', Fe2+, Fe3+ and other ions generally present in a cell cytoplasm or in an organelle lumen.
In one embodiment, the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endoplasmic reticulum vesicle, the compound C is Ca2+, and the membrane transport protein is a Sarco/endoplasmic reticulum Ca2+ ATPase (SER.CA) pump or the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endolysosome vesicle or extracellular lysosome vesicle, the compound C is K+, and the membrane transport protein is a TMEM175 ion channel, and preferably the extracellular organelle vesicle in the method according to the first, second, third, and fifth embodiments of the first object of the present invention is an extracellular endoplasmic reticulum vesicle, the compound C is Ca2+, and the membrane transport protein is a Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump.
In one embodiment, the extracellular organelle vesicle of type 1 (respectively the extracellular organelle vesicle of reference type) in the method according to the fourth embodiment of the first object of the present invention is an extracellular endoplasmic reticulum vesicle, the compound C is Ca2+, and the membrane transport protein is a Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump.
In the method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the extravesicular aqueous medium has preferably an osmolarity OSext ranging from 0.1 to 600 mOsm/L, and preferably from 0.1 to 100 mOsm/L, before the contacting step with said compound C (i.e. before contacting step ii), ii'), II'), b), B)).
The intravesicular aqueous medium has preferably an osmolarity OSint ranging from 0.1 to 600 mOsm/L, and more preferably from 0.1 to 100 mOsm/L, before the contacting step with said compound C (i.e. before contacting step ii), ii'), II', b), B)).
In the method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the contacting step (i.e. contacting step ii), ii'), II'), b), B)) and contact maintaining step (i.e. contact maintaining step iii), iii'), III'), d), C)) are preferably performed by mixing said compound C and said extracellular organelle vesicle or by alternating a flow rate of said compound C and a flow rate of said extracellular organelle vesicle by means of a microfluidic system.
In the method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the contacting step (i.e. contacting step ii), ii'), II'), b), B)) is preferably carried out with a solution (more preferably an aqueous solution) comprising said compound C at a molar concentration ranging from 0.001 pM to 300 mM, and preferably ranging from 0.01 pM to 10 mM.
In this embodiment, step ii) (respectively step ii), ii'), II'), b), B)) is performed by contacting a solution of said compound C with said extracellular organelle vesicle. The solution comprising said compound C can be an aqueous solution such as a buffer solution, preferably having an osmolarity between 0.1 mOsm/L to 600 mOsm/L, and more preferably having an osmolarity between 0.1 mOsm/L to 100 mOsm/L.
In particular, the solution comprising said compound C is different from a hydrophobic solution such as an oily phase.
In the method according to the first object of the present invention , the extracellular organelle vesicle (respectively the extracellular organelle vesicle of reference type) (respectively the extracellular organelle vesicle of type 1) has preferably a lumen, is bilayer-bounded and is not surrounded by plasma membrane of a hosting cell.
The method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments), further comprises during contacting step (i.e. contacting step ii), ii'), II'), b), B)) and contact maintaining step (i.e. contact maintaining step iii), iii'), III'), d), C)), at least one patch-clamp step so as to measure an electric current between the extracellular and intravesicular aqueous media.
In one embodiment of the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the method further comprises adding at least one fluorophore molecule reporting for the compound C to the intravesicular aqueous medium so that during contacting step (i.e. contacting step ii), ii'), III'), b), B)) and contact maintaining step (i.e. contact maintaining step iii), iii'), III'), d), C)) a volumic concentration of the compound C in the intravesicular aqueous medium is measured.
In one embodiment, the fluorophore molecule is added before the contacting step with compound C (i.e. before contacting step ii), ii'), II'), b), B)), such as for example on the cells before extraction of the organelles or after extraction. This fluorophore molecule is permeable to membranes so that it is already present in the extracellular organelle vesicle before the measurement process. During the measurement process, with the contact of the extracellular organelle vesicle and compound C, given the entry of compound C in the extracellular organelle vesicle, the fluorophore molecule becomes fluorescent because its fluorescence is proportional to the intravesicular concentration of compound C.
In one embodiment of the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the method further comprises adding at least one biological modulator (also called biomodulator) reporting for the membrane transport protein, preferably to the intravesicular aqueous medium.
The biological modulator favors or regulates the functioning of the membrane transport protein. The biological modulator is proper to each membrane transport protein. As an example, the biological modulator is ATP when the membrane transport protein is a Sarco/endoplasmic reticulum Ca2+ ATPase (SER.CA) pump.
Other examples of biological modulators can include GTP, UTP, CTP, binding proteins such as G proteins, AMPc, Phospholamban, calmodulin, protein kinase A, protein kinase C, protein kinase G, protein kinase D, protein phosphatases such as PPI or PP2A, CaMKII, phosphoinositides, Nitric Oxide, PLC, cAMP, and cGMP.
Said biological modulators are well-known by a person skilled in the art and the person skilled in the art can select the appropriate biological modulator depending on the membrane transport protein implemented in the method of the present invention.
In one embodiment, the biological modulator is added simultaneously with compound C (i.e. during contacting step ii), ii'), II'), b), B)).
In a preferred embodiment, step ii) (respectively step ii), ii'), II'), b), B)) is performed by contacting a solution comprising said compound C and said biological modulator, with said extracellular organelle vesicle.
The solution comprising said compound C and said biological modulator can be an aqueous solution such as a buffer solution, preferably having an osmolarity between 0.1 mOsm/L to 600 mOsm/L, and more preferably having an osmolarity between 0.1 mOsm/L to 100 mOsm/L.
In particular, the solution comprising said compound C and said biological modulator is different from a hydrophobic solution such as an oily phase.
In one embodiment, in the method according to the first object of the present invention (including first, second, third, and fifth embodiments; respectively including fourth embodiment), at least one of the contacting step or the contact maintaining step leads to morphological change(s) of said extracellular organelle vesicle, and preferably to a deflation of said extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contacting step (i.e. contacting step ii), II'), b), B); respectively ii')) followed by a reswelling of the extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contact maintaining step (i.e. contact maintaining step iii), III'), d), C); respectively iii')).
These morphological changes are indicative of a good transporting activity of said membrane transport protein with respect to compound C (i.e. healthy patient, normal expression, promoter effect of the candidate compound, presence/good specifity of the membrane transport protein and/or good response of the membrane transport protein).
In one embodiment, in the method according to the first object of the present invention (including first, second, third, and fifth embodiments, respectively including fourth embodiment), at least one of the contacting step or the contact maintaining step leads to morphological change(s) of said extracellular organelle vesicle, and preferably to a deflation of said extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contacting step (i.e. contacting step ii), II'), b), B), respectively ii')) followed by no reswelling of the extracellular organelle vesicle (respectively of said extracellular organelle vesicle of type 1) during the contact maintaining step (i.e. contact maintaining step iii), III'), d), C), respectively iii')).
These morphological changes are indicative of a bad transporting activity of said membrane transport protein with respect to compound C (i.e. non-healthy patient, abnormal expression, inhibitor effect of the candidate compound, absence/bad specifity of the membrane transport protein and/or bad response of the membrane transport protein).
In one embodiment of the first object of the present invention (including first, second, third, fourth, and fifth embodiments), the method further comprises before step i), a), A), I') or i '), a step of adding at least an amount of said compound C into the intravesicular aqueous medium of the extracellular organelle vesicle (loading step). When compound C is an ion, such step can be called an ion loading step. Thanks to this loading step, the extracellular organelle vesicle is loaded with compound C and in particular specific ions.
Said loading step can be carried out:
- by incubating the extracellular organelle vesicle in a compound C-rich medium (the transport membrane protein can make compound C enter the extracellular organelle vesicle) with ATP, GTP, CTP and other molecules that allow to charge compound C in extracellular organelle vesicle with energy, or - by subjecting the extracellular organelle vesicle to a sonicator bath, in a compound C-rich medium (to open the pores in the extracellular organelle vesicle and let the compound C in), or
- by passing the extracellular organelle vesicle through constrictions in a compound C-rich medium (to help penetrating compound C into the extracellular organelle vesicle), or
- by permeabilising the membrane with detergents or molecules that are removed prior to the method of the present invention.
The extracellular organelle vesicles used in the method according to the first object of the present invention
The method according to the first object of the present invention (including first, second, third, fourth, and fifth embodiments) implements extracellular organelle vesicles.
Interestingly, the extracellular organelle vesicles allow a fast quantification of drug interaction and/or effect of one or more compounds on transport membrane protein activity. They facilitate the method of the present invention compared to other techniques from the art which have to concentrate the fragments of organelles and then do some biochemistry quantification. The claimed method allow to study organelles interactions between cellular components in a new way and the role of intracellular proteins and lipids.
According to the present invention, the term "extracellular organelle vesicles" (also called "giant extracellular organelle vesicles" or GEOVs) means swollen bilayer- bounded organelles generated, extracted and collected from their hosting cells, as vesicles (with a lumen being bilayer-bounded), freed from the plasma membrane which surrounded it.
The extracellular organelle vesicles can be extracted from mammalian, plant and/or bacteria cells, and preferably extracted from mammalian cells.
Examples of such cells include COS-7 cells, HeLa cells, HEK cells, CHO cells, fibroblast cells, red blood cells, T-cells, neuroblastoma cells, stem cells, iPSC (induced Pluripotent Stem Cells) derived into cell types such iPSC derived neuronal stem cells, iPSC derived mesenchymal stem cells, iPSC derived monocytes stem cells, iPSC derived cardiomyocyte stem cells, iPSC derived microglia stem cells, iPSC derived myotubes stem cells, etc ... Said cells can be obtained from the market, from laboratories and/or from hospitals, and/or recovered from organoids, biopsies, tissues, organs and/or organisms, from healthy or diseased human patient origin.
Said extracellular organelle vesicles can have a mean surface-to-volume ratio S/V (surface divided by the volume of a geometric shape, spherical in most of the case) ranging from 3 pm’1 to 0.15 pm’1, preferably from 2 pm to 0.15 pm , more preferably from 1.5 pm-1 to 0.15 pm4, the most preferably between 1.2 pm4 to 0.15 pm4. It generally corresponds to a mean size ranging from about 2 to 40 pm, preferably from 3 to 20 pm, more preferably from 4 to 15 pm, the most preferably from 5 to 10 pm.
For example, said extracellular organelle vesicles have an increased surface- to-volume ratio which generally ranges from 1.2 pm4 (for a size > 5 pm) to 0.3 pm-1 (for a size < 20 pm).
Such extracellular organelle vesicles preferably derive from organelles from the group consisting of endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, multivesicular bodies. In particular, both membrane and lumen of such extracellular organelle vesicles are composed of proteins, lipids and metabolites. The total amount of biochemical material of such extracellular organelle vesicles can be a fraction of the total composition of the organelle from which they are produced. This fraction is, at least, larger than 0.01 %, preferably larger than 0.1 %, more preferably larger than 1 %, the most preferably larger than 10 %. Such extracellular organelle vesicles are produced having none, one, or several contacts with other extracellular organelle vesicles. Such extracellular organelle vesicles are produced having none, one, or several contacts with cell components such as cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, plasma membrane.
In one preferred embodiment, said extracellular organelle vesicles are defined by at least one of the following features, and preferably all the following features:
- said extracellular organelle vesicles have a luminal volume which is bilayer- bounded; have a surface-to-volume at least lower than 3 pm4, 2 pm4, 1.5 pm4, 1.2 pm4, 1 pm4, 0.85 pm4, 0.75 pm4, 0.66 pm4, 0.6 pm4, 0.5 pm4, 0.42 pm4, 0.38 pm-1, 0.33 pm’1, 0.28 pm’1, 0.25 pm’1, 0.2 pm’1, 0.15 pm’1.
- said extracellular organelle vesicles are produced from organelles of the source cell encompassing endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosome, endosome, peroxisome, multivesicular bodies.
- said extracellular organelle vesicles are not surrounded by the plasma membrane of the source cell from which they are produced; meaning that said extracellular organelle vesicles are in an extracellular medium.
- said extracellular organelle vesicles have membrane and lumen composed of proteins, lipids and metabolites. This biochemical composition is a fraction of the total composition of the organelle from which the extracellular organelle vesicles are produced. This fraction is, at least, larger than 0.01 %, 0.1 %, 0.5 %, 1 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 90 %, 100 %.
- said extracellular organelle vesicles have a volume at least larger than 5 pm3, 15 pm3, 30 pm3, 60 pm3, 100 pm3, 180 pm3, 260 pm3, 380 pm3, 520 pm3, 1000 pm3, 1500 pm3, 2000 pm3, 3000 pm3, 4500 pm3.
- said extracellular organelle vesicles have a size at least larger than 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 18 pm, 20 pm in all spatial directions.
- said extracellular organelle vesicles have none, one, or several contacts with other GEOVs.
- said extracellular organelle vesicles have none, one, or several contacts with cell components such as cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, peroxisomes, plasma membrane and other organelles.
Said extracellular organelles vesicles can be functional further characterized in that said GEOVs transport neutral species and ions (membrane transport protein activity that is ranging from at least 0.1 %, 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % of that of the membrane transport activity in a reference cell, e.g., the source cell); comprise protein activity that is ranging from at least 0.1 %, 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 % of that of the membrane transport activity in a reference cell, e.g., the source cell. The extracellular organelle vesicles can be stored, and later delivered for research purposes. The extracellular vesicles are stable at a temperature of less than 4°C, or -20°C, or -80°C for at least 1, 2, 3, 6, or 12 hours; 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, or 4 weeks; 1, 2, 3, or 6 months; or 1, 2, 3, 4, or 5 years. Said extracellular organelle vesicles can be produced by a first or second process, said first and second processes being described below.
Such processes control lysis parameter to minimize organelle damages to recover highly functional organelles vesicles. In particular the second process is a reproducible process of extraction where each cell will be subjected to the same stress, being thus compatible with industrial requirements : volume production, organelle viability and reproducibility.
The first process to produce the extracellular organelle vesicles
The first process is able to generate, extract and recover stable and functional organelles from cells, i.e. to produce said extracellular organelle vesicles.
Extracellular organelle vesicles can be produced from any cells, according to a first process comprising at least the following steps: u) Contacting the cells during 0.5 to 30 minutes with an hypotonic aqueous medium with an osmolarity ranging from 0.1 to 100 mOsm/L (for swelling both cells and its intracellular bilayer-bounded organelles, disrupting cell both cytoskeleton and extracellular matrix, spherizing the cell); v) Applying a membrane tension on cells ranging from 10'3 to 5 mN/m, during 10'4 to 100 seconds (for lysis and the removal of cell plasma membranes); w) Recovery of extracellular organelle vesicles into the hypotonic aqueous medium.
According to a particular embodiment of the first process, the cells from which the extracellular organelle vesicles are obtained can be cultured on a support or in suspension in bulk in any appropriate medium well known from the art, or can be recovered from organoids, tissues, organs or organisms. Any type of cells (mammalian, plant, or bacteria cells) can be used to generate organelles according to the first process. For example, cells derived from Cos7, Huh, mammalian cells, human cells, tumor cells, obtained from the market, laboratories and hospital patients.
According to a particular embodiment of the first process, step u) is performed using a hypotonic aqueous medium which is any aqueous solution with an osmolarity ranging from 0.1 to 100 mOsm/L, preferably from 1 to 50 mOsm/L, more preferably from 5 to 50 mOsm/L, the most preferably from 10 to 40 mOsm/L, during 0.5, 3, 5, 7, 10, 15, 20 to 30 minutes, derived from, for example, buffer solutions (Diluted DPBS), diluted cell culture medium (DMEM), ionic solutions, salt solutions (e.g. CaC or KCI solutions), diluted buffer, water, etc..., for the generation of stable and functional giant organelle vesicles. Thus for example, for COS-7 cells, the cytoplasm osmolarity is around 300 mOsm/L, meaning that all kind of aqueous solutions with an osmolarity below about 100 mOsm/L and higher than 0.1 mOsm/L, can be used to generate giant organelle vesicles. Such hypotonic aqueous medium enables to instantly apply to cells and its intracellular organelles an adequate non-destructive, fast and effective osmotic shock to generate spherical swollen cells and enlarged organelle vesicles. Otherwise, the swelling protocols are too long and not effective, leading to non-spherical compartments and protein degradation, which makes the production of GEOVs impossible. The control of the swelling kinetics parameters can favor the production of giant extracellular organelle vesicles.
Step u) is a step of osmotic swelling to generate intracellular organelles having an increased surface-to-volume ratio compared to their original form.
The aqueous media is eventually combined with chemicals, to increase the size of intracellular organelle vesicles (bilayer-bounded compartments) leading to the formation of enlarged vesicle of a size distribution never reported in the prior literature, called giant (intracellular) organelle vesicles (GIOVs) and, to reduce the plasma membrane lysis tension value.
In particular, some added molecules (cytoskeleton disruptor such as nocodazole, navelbine, latrunculin A, latrunculin B, cytochalasin; kinesin, myosin and dinein motor inhibitors such as blebistatin, benzytoluen sulphonamide, butanediome monoxime) allow to disassemble the cytoskeleton and reduce the lysis tension of the cells. Such hypotonic aqueous medium ensures that in the majority of cases, the intracellular compartments will form, after cell lysis, giant extracellular spherical vesicles from the organelles with a size and surface-to-volume ratio never reached before by the processes of the prior art. Thus, the hypotonic medium of the first process can also contain one or more molecules to modulate the surface-to-volume ratio distribution of giant extracellular organelle vesicles while preventing their degradation and the value of surface tensions to lyse the plasma membrane (e.g. protease inhibitors, molecular motors inhibitors, organelle-cytoskeleton contact inhibitors, cytoskeleton disruptors, detergents). Some added molecules (e.g. ion channels modulator/blocker: thaspsigargin, caffein, benzothiasepin; Extra-cellular matrix distruptor such as trypsin; Protein transport inhibitors; Protein signaling inhibitors such as xelospongin; Chemical detergents such as Triton-X-100, octylglucoside, DDM, carboxylic acids) allow the cells to swell faster, and therefore to decrease the surface-to-volume ratio of produced giant extracellular organelle vesicles more quickly. Finally, the cell swelling kinetics is crucial not only to decrease the input energy to lyse the cell plasma membrane of step v) but also to control the size of the GEOVs produced at the end of step w).
According to a particular embodiment of the first process, the hypotonic aqueous medium comprises one or more molecules chosen from the group consisting of: nocodazole, latrunculins, trypsin, misakinolides, mycalolides, aplyronides, vinblastine, rotenone, swinholides, jasplakinolides, vincristine, demecolcine, cytochalasins, colchicine, vinca-alcaloids, dihydropyridine, phenylalkylamine, benzothiazepine, gabapentinoids, blebistatin, benzytoluen sulphonamide, butanediome monoxime, thaspsigargin, xelospongin, Triton X-100, Tween, SDS, Brij, Octyl Glucoside, octyl thioglucoside, CHAPS, CHAPSO, magnesium, said molecules being added before step u), at step u), u'), v) and/or w).
According to a particular embodiment of the first process, prior to the generation of giant extracellular organelle vesicles, cells may be treated with molecules to prevent protein degradation, modulate biochemical reactions on organelles, etc... so that giant extracellular organelle vesicles can bear specific biochemical properties. The metabolic conditions and the architecture of cells and its organelles can thus be further modified before step v), by chemicals and/or by modifying the expression level of proteins impacting the architecture of cells and/or their organelles and/or the metabolic conditions. Indeed prior to step u), cells can be treated with chemicals or can be modified to overexpress or repress the level of some proteins that impact the architecture of the cell and its organelles - notably the surface-to-volume ratio and the relative positioning of the organelles with each other - and the metabolic conditions. These manipulations enable controlling the size of the recovered giant extracellular organelle vesicles and their contact: tuning the surface- to-volume ratio, prior to the osmotic swelling, impacting the future recovered giant organelle size, with adapted osmotic swelling. For example, prior to inducing the osmotic swelling, the surface-to-volume ratio of the future produced giant extracellular organelles vesicles can be modulated (1) by altering the expression levels of proteins impacting organelle shape and contact sites with other organelles, plasma membrane and cytoskeleton, (2) by treating cells with molecules that alter: both the cytoskeleton and molecular motors, organelle contacts sites (with other organelles, plasma membrane and cytoskeleton), molecules-transporting-proteins activity localized on plasma membranes and organelles, signaling protein activity (3) by altering cellular metabolic pathways impacting organelles number and shape, surface, (4) any treatment mediate change in organelle surface-to-volume ratio and inter-organelle contact. For example, overexpressing climp63 prior to the generation and recovery of giant extracellular organelles vesicles leads to larger giant extracellular organelles vesicles emanating from the endoplasmic reticulum with sizes larger than 30 pm. In the same way, overexpressing Mfn2 prior to the generation and recovery of giant extracellular organelles vesicles leads to larger giant extracellular organelles vesicles emanating from the mitochondria with surface-to- volume ratio smaller than 0.75 pm-1. Pre-treating cells with nocodazole (or Latrunculin A) between 1 and 90 min before swelling allows the formation of bigger giant organelles from the ER. Adding rapamycin between 12 to 24 hours before swelling formation allows to form bigger giant extracellular organelles vesicles from endosome, lysosome, autolysosome and multivesicular body. Adding bafylomicin before swelling and extraction also allows to obtain more giant extracellular organelles vesicles coming from autophagosomes.
According to a particular embodiment of the first process, said first process further comprises, after step u) and before step v), a step u') comprising generating a back-and-forth motion of the hypotonic aqueous medium to displace cells at a speed ranging from about 0.01 m/s to 10 m/s during about 0.01 seconds to 10 minutes, to disrupt both cytoskeleton and extracellular matrix of cells. This optional step u') applied to swollen cells of step u) thus allows to further apply a less important stretching membrane tension in step v) to lyse cells, open them completely and release GEOVs without lysing them. This is because the cytoskeleton gives the plasma membrane of the cell an additional resistance. Thus, when swollen cells and organelles from step u) are not subjected to such a motion, the average lysis tension needed in step v) is about ~7 mN/m (some cells lyse at under ~1 mN/m and other at ~10 mN/m, as shown in example), whereas for swollen cells and organelles from step u) subjected to such motion in step u'), the average lysis tension needed in step v) is about ~2 mN/m (A large majority of cells lyses at tension under 1 mN/m, as shown in example). Therefore, subjecting the cells to motion, without lysing them, allows to lower the further lysis tension without damaging the giant (intracellular) organelle vesicles still inside. In the literature, membrane rupture is almost always associated with cell lysis. In the first process, cell lysis leads to the release of all or almost all of the cell's contents (including organelles) into the extracellular medium. The state of the art does not show what happens after membrane rupture and misuse the term cell lysis because there is no release of intracellular compounds. Indeed, it is extremely common that plasma membrane rupture leads to vesiculation or to the opening of a bilayer pore which closes. In these cases, the organelles are never released although the state of the art is mentioning the term of plasma membrane rupture and/or cell lysis measurement. This is not because there is membrane rupture and/or cell lysis, that the intracellular content is released. The first process allows to drastically decrease (< 2 mN/m) the membrane tension to reached cell lysis, the removal the whole plasma membrane and the release of all or almost all GEOVs from a cell, without lysing them thanks to gentle lysis.
Accordingly, this step u') where a back-and-forth motion of the hypotonic aqueous medium is generated to displace the cells at a speed ranging from 0.01 m/s to 10 m/s during 0.01 seconds to 10 minutes promotes the disruption of both the cytoskeleton and the extracellular matrix of the cells, in order to reduce the plasma membrane lysis tension and provoke plasma membrane full opening following lysis.
According to a particular embodiment of the first process, step v) is performed to apply a stretching membrane tension (bilayer tension) on cells ranging from 10‘3 to 5 mN/m, preferably from 5.10‘3 to 4 mN/m, more preferably from 10‘2 to 2 mN/m, even more preferably from 10‘2 to 1 mN/m most preferably between 5.10'2 mN/m to 0.75 mN/m, during 10‘4 to 100 seconds, so as to break cell plasma membranes rapidly while preserving the structure of giant (extracellular) organelle vesicles released from lysed cells in the hypotonic aqueous medium. Generally, the first process allows to lower the stretching membrane tension to values less than about ~2 mN/m to extract and recover GEOVs into the medium. While when the usual stretching membrane tensions known from the state of the art for lysing cells are on average higher than 5 mN/m, all the organelles are fragmented into extracellular nano-vesicles which release their content into the medium and are therefore no longer manipulate unlike claimed GEOVs. Step v) can be carried out by using: mechanical force (e.g. suction pressure, stretching, shearing or acoustic wave), chemical agents and/or detergents, electric field, or laser (light) excitation. For example, the mechanical force is applied with a micro-pipette (radius from about 0,5 to 20 pm) with a suction pressure (generally ranging from about 0.1 to 600 mbar and more preferably from about 5 to 200 mbar (where previous bulk techniques use pressure much larger than 1 bar) that generates the expected stretching membrane tension on cells and leads to the lyse of plasma membrane and giant extracellular organelle vesicles recovery (see example section). For example, after cell swelling and giant intracellular organelle vesicles formation according to step u) of the first process, applying acoustic field (pressure waves) ranging in the ultrasonic range (preferably from about 16 Khz to 1000 Khz, more preferably from about 20 Khz to 400 Khz) during few seconds (e.g. 0.1 to 100 seconds) allows to generate the expected stretching membrane tension on cells and leading to the lyse of plasma membrane and giant extracellular organelle vesicles recovery (see example section). For example, after cell swelling and giant intracellular organelle vesicles formation according to step u) of the first process, chemicals working as detergents can be added in a control manner to create the expected stretching membrane tension on cells that leads to the lyse of plasma membrane and giant extracellular organelle vesicles recovery, such as carboxylic acid (with concentrations sufficient to generate plasma membrane lysis), Triton X, Tween, SDS, Brij, Octyl Glucoside, octyl thioglucoside, CHAPS, CHAPSO and others. Using detergents modifies the properties of the giant organelles.
Accordingly, said step v) where cells' plasma membrane tension is increased mechanically from cells of step u) or steps u) + u') leads to the lysis and full removal of their plasma membrane and the release in the hypotonic medium of stable and functional giant extracellular organelle vesicles (GEOVs), without lysing them.
To sum up, in the whole first process, the strong osmotic shock allows generate giant intracellular vesicles and to specifically destabilize the plasma membrane of cells, which then only needs a small mechanical perturbation to fully break and release the giant extracellular organelle vesicles, which are not broken thanks to the first process we developed for the plasma membrane lysis and removal.
The giant extracellular organelle vesicles of the first process make it now possible to turn toward applications previously unthinkable for industrial players. In addition, contact between such giant organelle vesicles can be conserved. Another advantage is the rapid isolation of giant organelle vesicles easily imaged with a microscope (e.g. about 5 to 20 pm sized).
Such giant extracellular organelle vesicles are functional, can be labelled, have any protein wanted on them, and can be easily picked and manipulated.
According to the first process, giant extracellular organelle vesicles can be recovered from step w) without any labeling and sorted out later by using several non-invasive methods including optical, morphological, mechanical approaches. However according to a particular embodiment of the first process, the cells of step u) can comprise cells transfected with at least one organelle protein marker or receiving molecules reporting for organelles to facilitate organelle identification. For example, the cells are transfected with at least one organelle protein tagged with a fluorescent or chemical marker used before or after swelling. For example, KDEL marks the ER. lumen, Tom20 marks mitochondria, LC3B marks autophagosomes, Lampl marks lysosomes and Golgi7 marks the Golgi apparatus. The extracellular organelle vesicles of the first process can be recovered with any type of protein of interest, in contact with different cellular components, or other giant extracellular organelle vesicles.
The second process to produce the extracellular organelle vesicles
The extracellular organelle vesicles can be obtained according to a second process implementing a device for recovering organelle vesicles from cells, said device comprising an extraction unit configured to extract organelle vesicles from cells, said process comprising a step of extraction of organelle vesicles from cells with said extraction unit, said extraction unit comprising a microfluidic chip having one or more microchannels configured to receive a fluid flow containing said cells, said microchannels comprising one or more constriction portions configured to apply mechanical constraints on said cells while passing therethrough.
The second process makes it possible to produce large quantities of organelle vesicles to make purified and concentrated samples that reach the market. The microfluidic extraction is ideal to reach flow rates of typically a million of large organelle vesicles extracted per hour continuously. Moreover, the high frequency extraction unit of the invention can be directly connected to a sorting machine.
The device for recovering organelle vesicles from cells, comprises an extraction unit configured to extract organelle vesicles from cells, said extraction unit comprising a microfluidic chip having one or more microchannels configured to receive a fluid flow containing said cells, said microchannels comprising one or more constriction portions configured to apply mechanical constraints on said cells while passing therethrough.
Such a microfluidic chip makes it possible to extract organelle vesicles from cells at high frequency, being thus compatible with industrial requirements.
In one embodiment, at least one of said constriction portions is connected to an upstream portion and/or a downstream portion of a same microchannel.
Said upstream portion preferably has a section which decreases, preferably monotonically, from an upstream part of the microchannel to said constriction portion.
Said downstream portion preferably has a section which increases, preferably monotonically, from said constriction portion to a downstream part of the microchannel.
In one embodiment, said upstream portion is formed by: - at least one first wall member defining with a transverse plane an angle ranging from 91° to 179°, preferably from 99° to 170°, for instance 105° or 130°, and/or
- at least one second wall member defining with said transverse plane an angle ranging from 90° to 170°, preferably from 90° to 160°, for instance 95° or 120°.
In one embodiment, said downstream portion is formed by:
- at least one first wall member defining with a transverse plane an angle ranging from 91° to 180°, preferably from 120° to 179°, for instance 150° or 170°, and/or
- at least one second wall member defining with said transverse plane an angle ranging from 90° to 170°, preferably from 90° to 160°, for instance 95° or 120.
When a constriction portion is connected to both an upstream portion and a downstream portion, it is preferred that said upstream portion is longer than said downstream portion, in relation to the direction of fluid flow.
In one embodiment, said constriction portion has:
- a section ranging from 100 pm2 to 2000 pm2, preferably from 170 pm2 to 650 pm2, for instance 225 pm2 or 330 pm2, and/or
- a width ranging from 4 pm to 15 pm, preferably from 7 pm to 13 pm, for instance 8, 9, 10, 11 or 12 pm, and/or
- a height ranging from 5 pm to 100 pm, preferably from 10 pm to 30 pm, for instance 25, 30, 35 or 40 pm, and/or
- a length ranging from 1 pm to 200 pm, preferably from 10 pm to 70 pm, preferably from 25 pm to 50 pm, for instance 20, 30, 40, 50 or 60 pm, and/or.
At least one of said constriction portions may have a polygonal cross-section, for instance a rectangular or trapezoidal cross-section.
In one embodiment, at least one of said microchannels comprises several of said constriction portions, for example two or more constriction portions.
In an embodiment, the constriction section is decreased from one to another constriction portion of a same microchannel, in relation to the fluid flow direction. In one embodiment, several of said microchannels comprises one or more of said constriction portions, respectively.
In one embodiment, the device implemented in the second process further comprises a preparation unit configured to prepare said cells.
In such embodiment, the process further comprises, before the extraction step, a step of preparing said cells with said preparation unit.
In one embodiment, said preparation unit comprises a means to put the cells in contact with a hypotonic aqueous medium.
In this embodiment, the cells are put in contact with said hypotonic aqueous medium.
The hypotonic aqueous medium is as defined for the first process described above.
In one embodiment, the device implemented in the second process further comprises a collecting unit configured to collect and/or to sort said organelle vesicles extracted from cells with said extraction unit.
In such embodiment, the process comprises, after the extraction step, a step of collecting said organelle vesicles with said collecting unit.
In one embodiment, said collecting unit is formed by a part of said microfluidic chip and/or includes an additional microfluidic chip.
In one embodiment, the device implemented in the second process further comprises an observation unit configured to observe said organelle vesicles extracted from cells with said extraction unit.
In such embodiment, the process further comprises, during and/or after the step of extraction, a step of observation of said organelle vesicles with said observation unit.
In one embodiment, said observation unit includes a camera and/or a microscope, for example a confocal microscope.
In one embodiment, the second process comprises an introduction of a fluid containing said cells within said microfluidic chip microchannels of said extraction unit with a flow rate ranging from 1 pb/min to 500 pb/min, preferably from 50 pL/min to 200 pL/min, for instance 75, 100, 125, 150 or 175 pb/min.
Of course, the above embodiments can be combined with each other. Indeed, in the second process, the extraction step in the extraction unit corresponds to step v) of said first process defined above, the preparation step of the cells in the preparation unit corresponds to step u) of said first process defined above, and the collecting step in the collecting unit corresponds to step w) of said first process defined above.
The invention relates as a second object to a kit for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein with respect to at least one compound C, wherein it comprises:
- at least one extracellular organelle vesicle,
- at least one compound C, wherein said membrane transport protein has a transporting activity with respect to said compound C, and
- optionally instructions.
The extracellular organelle vesicle, the membrane transport protein and compound C are as defined in the second embodiment of the method according to the first object of the present invention.
The kit can optionally comprise a biological modulator reporting for the membrane transport protein, said biological modulator being as defined in the method according to the first object of the present invention.
The present invention is illustrated in more detail in the examples below, but it is not limited to said examples.
Examples
Prior to extracellular organelle vesicles production
Cell culture
Cos7, HeLa, HEK and fibroblast cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Before the extracellular organelle vesicles production protocol, cells were cultivated 48h in DMEM media at 37°C with 5% CO2. Cells were cultured left adhering in Matek dishes, pre-treated or not with adhesion agents.
Cell transfection reporting for organelle vesicles
Cells were transfected 24h with different plasmids fused with fluorescent protein markers (e.g., RFP for Red Fluorescent Protein marker or BFP for Blue Fluorescent Protein marker), so as to probe different organelles before the extracellular organelle vesicles production. These plasmids serve to express proteins reporting for different organelles. Kdel-RFP or Erox-BFP and Mito-BFP plasmids were used to identify the endoplasmic reticulum and the mitochondria respectively.
Extracellular organelle vesicles production
After cell culture and transfection, cells were brought into contact with an hypotonic aqueous medium as detailed below.
An initial HKM buffer was prepared with 50 mM Hepes (known as 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid), 120 mM potassium acetate, and 1 mM MgC (in Milli-Q water) at pH 7.4. Said initial HKM buffer has an osmolarity of 280 ± 15 mOsm/L.
The initial HKM buffer was diluted with H2O, pH 7.4, at 37°C and 5% CO2 to reach an osmolarity of 20 mOsm/L and form a diluted HKM buffer (hypotonic aqueous medium).
Then, before the cells were confluent, the DMEM medium is removed and the cells are brought into contact with the diluted HKM buffer during 15 minutes.
A back-and-forth motion of the hypotonic aqueous medium is then generated to displace cells during 5 seconds. The back-and-forth motion is performed on all the volume of the hypotonic aqueous medium 3 times in a row. This step allows to destabilize the cytoskeleton of the cell and to reduce its lysis tension. At that step, organelle vesicles are still within the cells (i.e. intracellular organelle vesicles).
Then, a membrane tension is applied on cells so as to produce extracellular organelle vesicles without lysing them. At that step, the extracellular organelle vesicles are released (or produced) thanks to the lysis and the removal of the plasma membrane. The membrane tension is carried out by micro-manipulation of the cells under confocal microscope thanks to a micropipette aspiration technique. Micropipette radius is around 1 pm. Thanks to a slight aspiration, a bilayer tongue of the plasma membrane was sucked into the micropipette. The aspiration is then increased at an approximate rate of 10 mbar/min, causing a proportional increase in the bilayer surface tension. At a certain applied tension (e.g. surface tension < 0,75mN/m), the plasma membrane ruptured thanks to a pore opening in the plasma membrane and the extracellular organelle vesicles are released (or extracted).
After extraction, one or more extracellular organelle vesicles are isolated using a micropipette. The micropipettes are used to hold a vesicle during the following methods of the present invention. Before the addition of molecules (ions, sugars or drug molecules), an acquisition is launched to observe the initial size of the vesicle.
Before the addition of molecules (ions, sugars or drug molecules), an acquisition is launched to observe the initial size of the extracellular organelle vesicle.
Visualizing extracellular organelle vesicles under confocal microscopy
Once formed and extracted, the extracellular organelle vesicles are recovered and observed under a confocal microscope. Extracellular organelle vesicles can be observed thanks to the different above-mentioned markers (RFP) with a LSM800 Zeiss confocal microscope.
"Patchability" with micropipettes
Micropipettes were made from capillaries (1.0 OD x 0.58 ID x 150 Lmm 30- 0017 GC100-15b; Harvard Apparatus) with a micropipette puller (model P-2000; Sutter Instruments). Micromanipulation was performed with TransferMan 4r (Eppendorf). Pressure measurement unit (DP103) was provided by Validyne Engineering.
Example 1: method for examining the transporting activity of Ca2+ pump as membrane transport protein of the Endoplasmic Reticulum
Once the acquisition is started, the extracellular organelle vesicle, held by the micropipette is passed through an aqueous solution containing CaC (60 mOsm/L) and ATP (100 pM). Contacting calcium ions with said extracellular organelle vesicle increases the osmolarity of the extravesicular aqueous medium. Due to the difference in internal osmolarity of the vesicle (20 mOsm/L) and the CaC solution (60 mOsm/L), the vesicle deflates in a few seconds linked to an external water flow. In the following minutes, a re-swelling process is gradually observed. This innovative process allows to examine the transporting activity of the ion pump SERCA localized on Endoplasmic Reticulum vesicles.
Figure 1 A) represents an extracellular organelle vesicle of endoplasmic reticulum (ER) which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L) as an extravesicular aqueous medium. This extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), over-expressed by COS7 cells, before organelle vesicles extraction.
The ER extracellular organelle vesicle exhibits both its volume and fluorescence signal (respectively noted Vi and Fi) at initial state. At t = Os, CaCh (60 mOsm/L) and ATP (100 |jM) were added. At t = 20s, the ER. extracellular organelle vesicle is deflated : it reached its minimum volume (Vii) and its associated maximum fluorescence signal (Fii).
Then it re-swells to reach its final volume and fluorescence signal ( f and Ff) (t = 90s). Dotted line circles all correspond to the size of the ER extracellular organelle vesicle at the maximum deflated state (Vii).
Figure 1 B) represents plot of the mean vesicle fluorescence signal (F/Fi) and relative volume variations of vesicle V/Vi (measured in brightfield) and the associated opposite ratio (Vi/V). (F/Fi) and (Vi/V) are highly correlated and seem proportional. The variation of vesicle fluorescence signal is inversely proportional to the variation of vesicle volume. At t = 100s, the vesicle has almost reached its initial state of swelling.
By contrast, when the same experiment as above is performed with sucrose instead on calcium ions, no re-swelling is observed as detailed below.
Once the acquisition is started, the ER extracellular organelle vesicle, held by the micropipette is passed through an aqueous solution containing sucrose (60 mOsm). Due to the difference in internal osmolarity of the ER extracellular organelle vesicle (20 mOsm/L) and the sucrose solution (60 mOsm/L), the ER extracellular organelle vesicle deflates in a few seconds linked to an external water flow. However, in this case no re-swelling process is visible as shown in figure 2. Sucrose is thus not transported inside the ER extracellular organelle vesicle as it was the case of calcium ions with SERCA. Sucrose can thus be used as a control or reference in the method of the present invention.
Thus, we can define a method for examining the transporting activity of intracellular ion channel/pump activity based on parameters changes such as volume and/or fluorescence changes of extracellular organelle vesicles, that can also be combined with other parameters changes.
Accordingly, the percentage of relative volume variation during the re-swelling phase which is followed under the microscope can give the relative activity of an ion channel. All parameters observable during the method of the present invention allow to obtain information on the activity of membrane transport proteins present on organelle vesicles. The relative volume changes, the kinetics of volume change, the initial slopes of change, the values of the ratio between the initial volume and the volume after deflation and before re-inflation and/or of the ratio between the volume after deflation and before re-inflation and the final volume after re-inflation are relevant parameters to examine the transporting activity of a membrane transport protein.
Example 1 demonstrates applicability of the method according to the first object of the present invention, first embodiment. In particular, figures 1 and 2 clearly show the transporting activity of the SER.CA ion pump as membrane transporting protein with respect to Ca2+ as compound C with f/ ii > Vf,ref/Vii,ref, and f/ i > Vf,ref/Vi,ref, where sucrose is used instead of Ca2+ as the reference compound.
Exemple 2: method for diagnosing a disease affecting a subject, said disease being characterized by abnormal expression and/or abnormal activity of Ca2+ pump as a membrane transport protein of the Endoplasmic Reticulum (first object of the present invention, third embodiment)
Figure 3 A) represents an extracellular organelle vesicle of endoplasmic reticulum (ER) which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L) as an extravesicular aqueous medium. This extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), over-expressed by COS7 cells, before organelle vesicles extraction.
The ER extracellular organelle vesicle exhibits both its volume and fluorescence signal (respectively noted Vi and Fi) at initial state.
At t = Os, CaC (60 mOsm/L) and ATP (100 pM) were added. At t = 20s, the ER extracellular organelle vesicle is deflated : it reached its minimum volume (Vii) and its associated maximum fluorescence signal (Fii).
Then it re-swells to reach its final volume and fluorescence signal (Vf and Ff) (t = 90s). Dotted line circles all correspond to the size of the ER extracellular organelle vesicle before the beginning of the method (initial state).
Figure 3 B) represents plot of the mean vesicle fluorescence signal (F/Fi). At t = 300s, the ER extracellular organelle vesicle vesicle has reached a higher volume than the initial state, demonstrating an abnormal over-expression and/or abnormal activity of Ca2+ pump of the Endoplasmic Reticulum.
Example 3: other parameters that can be used in the methods of the present invention
Figure 4 represents an ER extracellular organelle vesicle which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L). This ER extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), overexpressed by COS7 cells, before organelle vesicles extraction. At t = 0s, CaC (60 mOsm/L) and ATP (100 |jM) were added. Both fluorescence (figure 4, top figures) and brigh field confocal microscopy (figure 4, bottom figures) snaps are visualized. The series of bright field images show that during deflation and re-inflation, the vesicle remains patched with the micropipette, which makes it possible to measure the current in whole cell patch clamp conformation generated by the activity of ion pumps of the ER. extracellular organelle vesicle. Thus, the membrane transport activity such as ion pump activity can be recorded using several simultaneous reading parameters:
- current parameter: as it can be seen in figure 4, the extracellular organelle vesicles are caught with micropipettes that are commonly used in patchclamp experiments. The patchclamp is an electrophysiology method that allows recording the activity of ion channels by recording currents. During the deflation-reinflation process, the extracellular organelle vesicles remain patched on the micropipettes in a conformation identical to the so called "cell-attached conformation" during a typical patch clamp experiment. It is therefore possible to record the activity of the ion pump on extracellular organelle vesicles while simultaneously observing the variation in current (recorded in patch clamp) and size (recorded in patch clamp).
- fluorescence parameter: the resident fluorescent proteins of the extracellular organelle vesicle are not permeable to its membrane. Thus they cannot cross the membrane during the swelling process. When the extracellular organelle vesicle deflates, the fluorescent signal (related to KDEL-RFP or ERox-BFP) increases, because the volume concentration of fluorescent proteins is higher. When the extracellular organelle vesicle is reinflated, the concentration of fluorescent protein decreases again, and thus the fluorescence signal as well.
Accordingly, extracellular organelle vesicles are « patchable » with a micropipette during the deflating-reswelling process.
Figure 5 represents an ER extracellular organelle vesicle which is maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L). This extracellular organelle vesicle has been isolated thanks to an ER resident protein (KDEL), overexpressed by COS7 cells, before organelle vesicles extraction. Before organelle extraction, cells were incubated during lh with a fluorophore called "Fluo8-AM" to report for calcium volumic concentration in organelles thanks to green fluorescence emission.
At t = Os, CaC (30 mOsm/L) and ATP (100 pM) were added. Fluorescence microscopy snaps are visualized on figure 5 and show calcium concentration inside the ER. extracellular organelle vesicle during the experiment. Fluorescence signal is rising after addition of CaC , showing that Ca2+ is internalized in the ER extracellular organelle vesicle during the experiment.
Other fluorophores than "Fluo8-AM" such as "Fluo3-AM" or "Fura-2" fluorophores can also be used to follow directly Ca2+ concentrations variation inside ER extracellular organelle vesicles. These fluorophores are known to emit fluorescence that is proportional to Ca2+ volumic concentration in a compartment.
Membrane surface tension parameter can be another way of examining the activity of a membrane transport protein: when the vesicle swells, its volume increases, as well as the area of its membrane that delimits it. This results in an increase in the surface tension of the membrane of the extracellular organelle vesicle which can be calculated simply with a micropipette and with the laws of Laplace. The addition of fluorescent molecules such as "Flipper-TR" can be used to visualize a variation in the surface tension of the extracellular organelle vesicle membrane linked to the activity of ion channels causing the re-swelling.
Example 4: method for screening of candidate compounds intended to modulate the activity of Ca2+ pump as a membrane transport protein of the Endoplasmic Reticulum (first object of the present invention, second embodiment)
Once the ER extracellular organelle vesicles are extracted, thapsigargin (10 pM) in DMSO, is added to the medium. Thapsigargin is a known inhibitor of the SERCA pump. After 10 minutes of incubation with thapsigargin, and once the acquisition is started, the extracellular organelle vesicle, held by the micropipette is passed through an aqueous solution containing CaC (60 mOsm/L) and ATP (ImM). Due to the difference in internal osmolarity of the extracellular organelle vesicle (20 mOsm/L) and the CaC solution (60 mOsm/L), the vesicle deflates in a few seconds linked to an external water flow. Surprisingly, the reswelling process is decreased or not present compared to the same experiment performed on ER extracellular organelle vesicle not incubated with the thapsigargin.
Figure 6 A) represents an ER extracellular organelle vesicle maintained thanks to a micropipette into a HKM diluted buffer (20 mOsm/L). This extracellular organelle vesicle has been isolated thanks to an ER resident protein, over-expressed by COS7 cells, before organelle vesicles extraction. This extracellular organelle vesicle was incubated with thapsigargin at 10 pM during 10 min. At t = Os, CaCh (60 mOsm/L) and ATP (100 |jM) were added. Dotted line circles all correspond to the size of the extracellular organelle vesicle before the beginning of the process.
Figure 6 B) represents plot of the mean extracellular organelle vesicle fluorescence signal ( F/Fi) where corresponding volume of the vesicles are shown. This plot shows that, in presence of thaspigargin, the re-swelling process is almost off.
Example 4 demonstrates applicability of the method according to the first object of the present invention, second embodiment. In particular, figure 1 and 6 clearly show the inhibitory effect of thapsigargin on the transporting activity of the SERCA ion pump as membrane transporting protein with respect to Ca2+ as compound C with f/ ii < Vf,ref/Vii,ref, and f/ i < Vf,ref/Vi,ref, where the reference ratio is obtained without the presence of thapsigargin.
Example 5: method for determining the specificity of the SERCA pump as a membrane transport protein with respect to an extracellular organelle vesicle of endoplasmic reticulum type or the residency of the SERCA pump as a membrane transport protein in an extracellular organelle vesicle of endoplasmic reticulum type (first object of the present invention, fourth embodiment)
In this example, an Endoplasmic Reticulum extracellular organelle vesicle and a mitochondria extracellular organelle vesicle are isolated simultaneously with micropipettes, and visualized. ER and mitochondria extracellular organelle vesicles were both put in contact with CaC (30 mOsm/L) and ATP (100 pM) during one hour.
Figure 7 represents Endoplasmic Reticulum extracellular organelle vesicle and mitochondria extracellular organelle vesicle are maintained thanks two micropipettes into a HKM diluted buffer (30 mOsm/L). These extracellular organelle vesicles were isolated thanks to ER and mitochondria resident proteins, over-expressed by COS7 cells, before organelle vesicles extraction.
At t = 0s, CaC (30 mOsm/L) and ATP (100 pM) were added. Dotted line circles all correspond to the size of the vesicle before contacting with CaC . lh after contacting with CaCb, the ER extracellular organelle vesicle volume exhibits the same volume as its initial one, whereas the mitochondria extracellular organelle vesicle has deflated.
Thanks to the method of the present invention, it is possible to demonstrate that the SERCA pump is specific to the ER organelle vesicle. Example 6: method for evaluating the response of the SERCA pump as a membrane transport protein contained in a membrane of an extracellular organelle vesicle of endoplasmic reticulum type with respect to a response of said membrane transport protein contained in a nucleus cell component (first object of the present invention, fifth embodiment)
In this example, an Endoplasmic Reticulum extracellular organelle vesicle and a nucleus cell component are isolated together from a same cell with one micropipette, and visualized. ER extracellular organelle vesicle and nucleus cell component were both put in contact with CaC (20 mOsm/L) and ATP (100 pM) during 5 minutes.
Figure 8 represents Endoplasmic Reticulum extracellular organelle vesicle and nucleus cell component are maintained thanks one micropipette into a HKM diluted buffer (30 mOsm/L). The extracellular organelle vesicle and the nucleus cell component were respectively isolated thanks to ER and nucleus probes, overexpressed by COS7 cells, before organelle vesicle and nucleus extraction.
At t = Os, CaC (60 mOsm/L) and ATP (100 pM) were added. Dotted line circle corresponds to the size of the endoplasmic reticulum vesicle before contacting with CaC . 5 min after contacting with CaCb, the ER extracellular organelle vesicle volume exhibits a bigger volume as its initial one, whereas the nucleus cell component has deflated with no re-swelling.
Thanks to the method of the present invention, it is possible to demonstrate that the SERCA pump has a better response when localized in the membrane of an ER organelle vesicle compared to the nucleus cell component.
Example 7: method for evaluating the response of the SERCA pump as a membrane transport protein contained in a membrane of an extracellular organelle vesicle of endoplasmic reticulum type with respect to a response of said membrane transport protein contained in a plasma membrane cell component (first object of the present invention, fifth embodiment)
In this example, an Endoplasmic Reticulum extracellular organelle vesicle and a plasma membrane cell component are isolated with micropipettes. In this example, an Endoplasmic Reticulum extracellular vesicle and a plasma membrane cell component do not come from the same cell.
The SERCA protein is over-expressed in cell 1 on its native organelle, the endoplasmic reticulum. In cell 2, SERCA is relocalized by genetic construction on the plasma membrane cell component. This finally serves to isolate with micropipettes, one endoplasmic reticulum extracellular vesicle with SER.CA extracted from cell 1 and one plasma membrane cell component enriched with SER.CA deriving from cell 2.
These extracellular organelle vesicle and plasma membrane cell component were isolated thanks to ER. and plasma membrane protein probes, over-expressed by COS7 cells, before organelle vesicle and plasma membrane cell component extraction.
ER extracellular vesicle and plasma membrane cell component were both put in contact with CaC (60 mOsm/L) and ATP (100 pM) during 5 minutes.
At t = Os, CaC (60 mOsm/L) and ATP (100 pM) were added. 5 min after contacting with CaC , the ER extracellular organelle vesicle volume exhibits a bigger volume as its initial one, whereas the plasma membrane cell component has deflated (figure not shown).
Thanks to the method of the present invention, it is possible to demonstrate that the method of the present invention has a much better response than a well- known method which implements relocalization of the SERCA pump into the plasma membrane by genetic construction.
Example 8: method for examining the transporting activity of K+ ion channel TMEM175 as membrane transport protein of the lysosome and endolysosome and the efficacy of drug candidates to modulate the transporting activity of TMEM175.
Before starting acquisition, extracellular endolysosome vesicles are charged with K+ ions thanks to a bath sonicator and an extracellular solution (210 mOsm/L) containing 100 mM of KCI and 10 mM of Hepes, pH 7.2. Then, extracellular endolysosome vesicles are seeded on a glass plate for observation. Before starting acquisition, extravesicular aqueous solution is removed to be changed when starting acquisition.
Once the acquisition is started, the extracellular organelle vesicle, held by the micropipette is passed through an extra-vesicular aqueous solution (200 mOsm/L) containing 1 mM of KCI and 200mM of sucrose. Contacting very small amount of potassium ions with said extracellular organelle vesicle generates a concentration gradient of K+ between the inside and the outside of the vesicles. Due to the difference in internal K+ of the vesicle (100 mM) and K+ in the external solution (1 mM), the vesicle deflates in a few seconds linked to an external potassium flow. In the following minutes, a re-swelling process is not observed. This innovative process allows to examine the transporting activity of the ion channel TMEM175 localized on lysosomes or endolysosomes vesicles in absence and/or in presence of an antagonist (4-AP) and/or agonist (DCPIB).
Thus, we can define a method for examining the blocking/activating efficacy of an antagonist/agonist on the transporting activity of intracellular ion channel/pump activity based on parameters changes such as volume and/or fluorescence changes of extracellular organelle vesicles, that can also be combined with other parameters changes.
Scope of the process:
As advertised the methods of the present invention can be combined with a patch clamp measurement on extracellular organelle vesicles.
In case the concentration of ion channels or pumps is too low, it is possible to overexpress them 24-48 hours before in cell culture, before the extraction and purification of organelle vesicles and their activity measurement.
Thus, the methods of the present invention concern ion channels / ion pumps / transporters that are released and localized on extracted organelle vesicles : Endoplasmic reticulum, mitochondria, lysosome, Golgi apparatus, vacuole, endolysosome, autophagosome, autolysosomes, endosomes, multivesicular bodies or a complex of 2 or more organelles in contact.
The methods of the present invention can be used in the pharmaceutical market as :
- Drug candidate efficacy tests targeting intra-cellular ion channels/pumps for screening campaign in drug discovery phases (Hit discovery, Hit-To-Lead, Lead Optimization).
- Toxicity test of drug candidates in drug discovery or non-regulatory pre- clinical phase on organellar ion channel / pumps that could be implicated for heart arrythmia or failure, muscle contractions or others toxic implications on organs or the whole organism.
Diagnostic testing of certain pathologies (cancers, myopathies, neurodegenerative diseases or metabolic diseases) where intracellular ionic flow disorders are caused due to dysfunction or disruption of the activity of organelle transmembrane ion channels.
Figure 9 A) represents an extracellular organelle vesicle 1 provided in an extravesicular aqueous medium 2, said extracellular organelle vesicle 1 comprising a membrane 3 surrounding an intravesicular aqueous medium 4. A membrane transport protein 5 is contained in said membrane 3. Figure 9 B) represents the contacting step of said extracellular organelle vesicle 1 with a compound 6 (e.g. compound C as defined in the present invention) in the presence of a biomodulator 7 of the membrane transport protein 5. The membrane transport protein 5 has a transporting activity with respect to said compound 6.

Claims

1. An in vitro or ex vivo method for examining the transporting activity and/or studying the biological behaviour of at least one membrane transport protein (5) with respect to at least one compound C (6), wherein said method comprises at least the following steps: i) providing in an extravesicular aqueous medium (2) at least one extracellular organelle vesicle (1) comprising a membrane (3) surrounding an intravesicular aqueous medium (4), ii) contacting said compound C (6) with said extracellular organelle vesicle (1), and iii) maintaining said compound C (6) and said extracellular organelle vesicle (1) into contact, wherein said method further comprises:
- observing and/or measuring morphological change(s) of said extracellular organelle vesicle (1) between step i) and step iii) and/or between step ii) and step iii), said morphological change(s) resulting in a change in at least one parameter, preferably selected from a volume V (in pm3), a membrane surface tension T (in N/m), and a luminal fluorescence value F (in arbitrary unit),
- comparing said morphological change(s) of said extracellular organelle vesicle (1) with reference morphological change(s), wherein said membrane transport protein (5) is contained in at least one of said membrane (3) or a membrane of a reference extracellular organelle vesicle.
2. The method according to claim 1, wherein said method comprises at least the following steps: i) providing in an extravesicular aqueous medium (2) at least one extracellular organelle vesicle (1) comprising a membrane (3) surrounding an intravesicular aqueous medium (4), said membrane (3) containing said at least one membrane transport protein, and said extracellular organelle vesicle (1) being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii) contacting said compound C (6) with said extracellular organelle vesicle (1), and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a volume Vii (in |jm3), a membrane surface tension Tn, and a luminal fluorescence value Fii (in arbitrary unit), iii) maintaining said compound C (6) and said extracellular organelle vesicle (1) into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a final volume f (in pm3), a final membrane surface tension Tf, and a final luminal fluorescence value Ff (in arbitrary unit), iv) calculating at least one ratio selected from f/ ii, Tf/Tn, Ff/Fn, f/ i, Tf/Ti, and Ff/Fi, v) comparing said ratio with a corresponding reference ratio selected from Vf, ref/ ii, ref, Tf,ref/Til,ref, Ff,ref/Fil,ref, Vf, ref/ Vi, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref, SO 8S tO evaluate the transporting activity and/or the behaviour of said membrane transport protein (5) with respect to said compound C (6), wherein the corresponding reference ratio is obtained by applying said method with a reference compound instead of said compound C (6), said membrane transport protein (5) not having a transporting activity with respect to said reference compound or said membrane transport protein (5) having a known transporting activity with respect to said reference compound.
3. The method according to claim 1, wherein it is a method for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein (5) with respect to at least one compound C (6), wherein it comprises at least the following steps: a) providing in an extravesicular aqueous medium (2) at least one extracellular organelle vesicle (1) comprising a membrane (3) surrounding an intravesicular aqueous medium (4), said membrane (3) containing said at least one membrane transport protein (5), and said extracellular organelle vesicle (1) being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), b) contacting said compound C (6) with said extracellular organelle vesicle (1), and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit) of said extracellular organelle vesicle (1), wherein said membrane transport protein (5) has a transporting activity with respect to said compound C (6), c) contacting at least one candidate compound with said extracellular organelle vesicle (1), wherein step c) is carried out before step b) or after step b), and preferably before step b), d) maintaining said compound C (6), said candidate compound, and said extracellular organelle vesicle (1) into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit), e) calculating at least one ratio selected from f/ ii, Tf/Tii, Ff/Fu, f/ i, Tf/Ti, and Ff/Fi, f) comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf,ref/ i,ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the transporting activity and/or the behaviour of said membrane transport protein (5) with respect to compound C (6) in the presence of said candidate compound, wherein the corresponding reference ratio is obtained by applying said method without the presence of said candidate compound or by applying said method with a reference candidate compound instead of said candidate compound, said reference candidate compound having a known modulator effect on the transporting activity of said membrane transport protein (5) with respect to said compound C (6).
4. The method according to claim 3, wherein the contacting step c) is carried out with a solution comprising said candidate compound at a molar concentration ranging from 0.001 pM to 100 mM, and preferably ranging from 0.1 nM to 100 pM.
5. The method according to claim 3 or 4, wherein the contacting step c) is performed during at least 5 seconds, and preferably during at least 30 seconds.
6. The method according to any one of claims 3 to 5, wherein the method is carried out with extracellular organelle vesicles of different types so as to evaluate toxicity of said candidate compound with respect to some of said different types of extracellular organelle vesicles.
7. The method according to claim 1, wherein it is a method for determining abnormal expression and/or abnormal transporting activity of at least one membrane transport protein (5) with respect to at least one compound C (6), comprising at least the following steps:
A) providing in an extravesicular aqueous medium (2) at least one extracellular organelle vesicle (1) from genetically-modified cells or from cells of a patient, said extracellular organelle vesicle (1) comprising a membrane (3) surrounding an intravesicular aqueous medium (4), said membrane (3) containing said at least one membrane transport protein (5), and said extracellular organelle vesicle (1) being described by at least one parameter selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit),
B) contacting said compound C (6) with said extracellular organelle vesicle, (1) and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit),
C) maintaining said compound C (6) and said extracellular organelle vesicle (1) into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit),
D) calculating at least one ratio selected from f/ ii, Tf/Tii, Ff/Fii, f/ i, Tf/Ti, and Ff/Fi,
E) comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf, ref/ i, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the abnormal expression and/or abnormal transporting activity of said membrane transport protein (5) with respect to said compound C (6), wherein the corresponding reference ratio is obtained by applying said method to a reference extracellular organelle vesicle from cells of an healthy patient or from non-genetically-modified cells, and wherein said membrane transport protein (5) has transporting activity with respect to said compound C (6) in said cells of a healthy patient or in said non-genetically-modified cells.
8. The method according to claim 7, wherein said method is implemented to diagnose a disease affecting said patient, said disease being characterized by abnormal expression and/or abnormal activity of said at least one membrane transport protein (5) with respect to said compound C (6).
9. The method according to claim 1, wherein it is a method to evaluate the response of at least one membrane transport protein (5) contained in a membrane
Figure imgf000054_0001
membrane transport protein (5) contained in a cell component, wherein said method comprises at least the following steps:
I') providing in an extravesicular aqueous medium (2) at least said extracellular organelle vesicle (1), said extracellular organelle vesicle (1) comprising said membrane (3) surrounding an intravesicular aqueous medium (4), said extracellular organelle vesicle (1) being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit),
II') contacting at least one compound C (6) with said extracellular organelle vesicle (1), and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a volume ii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit), wherein said at least one membrane transport protein (5) has a transporting activity with respect to said compound C (6),
III') maintaining said compound C (6) and said extracellular organelle vesicle (1) into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle (1) selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit),
IV') calculating at least one ratio selected from Vf/Vii, Tf/Tii, Ff/Fu, Vf/Vi, Tf/Ti, and Ff/Fi,
V') comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff,ref/Fil,ref, Vf, ref/ Vi, ref, Tf,ref/Ti,ref, a nd Ff,ref/Fi,ref, wherein the corresponding reference ratio is obtained:
* by applying said method [i.e. steps I') to IV')] with a cell component instead of said extracellular organelle vesicle (1), or
* by further providing in said extravesicular aqueous medium (2) of said step I') a cell component in addition to said extracellular organelle vesicle (1).
10. The method according to claim 9, wherein the cell component is selected from cytosol biomolecules, microtubules, actin, filaments, cell nucleus, lipid droplets, ribosomes, centrioles, and plasma membrane.
11. The method according to any one of the preceding claims, wherein the extracellular organelle vesicle (1) is selected from organelles of endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, and multivesicular bodies.
12. The method according to claim 1, wherein it is a method to determine the presence of at least one membrane transport protein (5) in at least one extracellular organelle vesicle of type 1 (1), wherein said method comprises at least the following steps: i') providing in an extravesicular aqueous medium (2) at least one extracellular organelle vesicle of type 1 (1), said extracellular organelle vesicle of type 1 (1) comprising a membrane (3) surrounding an intravesicular aqueous medium (4), said extracellular organelle vesicle of type 1 (1) being described by at least one parameter related to a morphological change selected from an initial volume Vi (in pm3), an initial membrane surface tension Ti (in N/m), and an initial luminal fluorescence value Fi (in arbitrary unit), ii') contacting at least one compound C (6) with said extracellular organelle vesicle of type 1 (1), and optionally measuring at least one parameter related to a morphological change of said extracellular organelle vesicle of type 1 (1) selected from a volume Vii (in pm3), a membrane surface tension Tii (in N/m), and a luminal fluorescence value Fii (in arbitrary unit), wherein said at least one membrane transport protein (5) has a transporting activity with respect to said compound C (6),
Hi') maintaining said compound C (6) and said extracellular organelle vesicle of type 1 (1) into contact, and measuring at least one parameter related to a morphological change of said extracellular organelle vesicle of type 1 (1) selected from a final volume f (in pm3), a final membrane surface tension Tf (in N/m), and a final luminal fluorescence value Ff (in arbitrary unit), iv') calculating at least one ratio selected from f/ ii, Tf/Tii, Ff/Fu, f/ i, Tf/Ti, and Ff/Fi, v') comparing said ratio with a corresponding reference ratio selected from Vf,ref/Vil,ref, Tf,ref/Til,ref, Ff, ref/ Fi 1 , ref , Vf, ref/ i, ref, Tf,ref/Ti,ref, and Ff,ref/Fi,ref SO 8S tO evaluate the presence of said membrane transport protein (5) in said at least one extracellular organelle vesicle of type 1 (1), wherein the corresponding reference ratio is obtained:
* by applying said method with an extracellular organelle vesicle of a reference type instead of said extracellular organelle vesicle of type 1 (1), said extracellular organelle vesicle of a reference type comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein (5), or
* by further providing in said extravesicular aqueous medium (2) of said step i') an extracellular organelle vesicle of a reference type in addition to said extracellular organelle vesicle of type 1 (1), said extracellular organelle vesicle of a reference type comprising a membrane surrounding an intravesicular aqueous medium, said membrane containing said at least one membrane transport protein
(5).
13. The method according to claim 12, wherein said method is implemented with several types of extracellular organelle vesicles, preferably from a same type of cell, so as to screen which type(s) of organelle vesicles has said at least one membrane transport protein as a resident protein, said several types of extracellular organelle vesicles being preferably selected from the following types: endoplasmic reticulum, mitochondria, lysosome, endolysosome, Golgi apparatus, vacuole, chloroplast, autophagosome, autolysosomes, endosomes, peroxisome, multivesicular bodies.
14. The method according to any one of the preceding claims, wherein the membrane transport protein (5) is an ion channel, an ion pump, or a transporter, and preferably an ion channel or an ion pump.
15. The method according to according to any one of the preceding claims, wherein the compound C (6) is selected from ions, cellular metabolites, pyruvate, amino acids, fatty acids, fatty acids CoA, malate, citrate, isocitrate, and vitamins, and preferably selected from ions.
16. The method according to any one of the preceding claims, wherein the extravesicular aqueous medium (2) has an osmolarity OSext ranging from 0.1 to 600 mOsm/L, before the contacting step with said compound C (6).
17. The method according to any one of the preceding claims, wherein the contacting and contact maintaining steps are performed by mixing said compound C
(6) and said extracellular organelle vesicle (1) or by alternating a flow rate of said compound C (6) and a flow rate of said extracellular organelle vesicle (1) by means of a microfluidic system.
18. The method according to any one of the preceding claims, wherein the contacting step is carried out with a solution comprising said compound C (6) at a molar concentration ranging from 0.001 |jM to 300 mM, and preferably ranging from 0.01 pM to 10 mM.
19. The method according to any one of the preceding claims, wherein the extracellular organelle vesicle (1) has a lumen, is bilayer-bounded and is not surrounded by plasma membrane of a hosting cell.
20. The method according to any one of the preceding claims, wherein said method further comprises during contacting and contact maintaining steps, at least one patch-clamp step so as to measure an electric current between the extracellular and intravesicular aqueous media (2, 4).
21. The method according to any one of the preceding claims, wherein said method further comprises adding at least one fluorophore molecule reporting for the compound C (6) to the intravesicular aqueous medium (4) so that during contacting and contact maintaining steps a volumic concentration of the compound C (6) in the intravesicular aqueous medium (4) is measured.
22. The method according to any one of the preceding claims, wherein at least one of the contacting step or the contact maintaining step leads to morphological change(s) of said extracellular organelle vesicle (1), and preferably to a deflation of said extracellular organelle vesicle (1) during the contacting step followed by a reswelling of the extracellular organelle vesicle (1) during the contact maintaining step.
23. The method according to any one of the preceding claims, wherein said method further comprises before step i), a), A), I') or i'), a step of adding at least an amount of said compound C (6) into the intravesicular aqueous medium (4) of the extracellular organelle vesicle (1) or the extracellular organelle vesicle of type I (1).
24. A kit for screening of candidate compounds intended to modulate the transporting activity of at least one membrane transport protein (5) with respect to at least one compound C (6), wherein it comprises:
- at least one extracellular organelle vesicle (1),
- at least one compound C (6), wherein said membrane transport protein (5) has a transporting activity with respect to said compound C (6), and
- optionally instructions.
PCT/EP2024/060083 2023-04-19 2024-04-12 A method implementing morphological changes of extracellular organelle vesicles so as to examine the transporting activity of membrane transport proteins and other related methods Pending WO2024218019A1 (en)

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